Trends in Parasitology

Review

The Human Spleen in Malaria: Filter or Shelter?

Benoît Henry,1,2,3,4 Camille Roussel,1,2,3 Mario Carucci,1,2,3 Valentine Brousse,1,2,3
Papa Alioune Ndour,1,2,3 and Pierre Buffet1,2,3,4,*

The human spleen is an immune sentinel and controls red blood cell (RBC) qual- Highlights
ity. By mechanically retaining subsets of infected RBCs, the spleen may reduce In malaria-infected subjects the spleen
the pace at which the parasite biomass increases before the adaptive immune senses subtle mechanical changes in in-
fected and uninfected RBCs. This filter-
response operates. Conversely, the spleen may contribute to malaria pathogen-
ing function may regulate parasite
esis, particularly anemia that is associated with splenomegaly. Large spleens biomass and induce clinical signs of ma-
may also shelter parasites in chronic carriers. Upon treatment with artemisinins, laria, like splenomegaly and anemia.
the spleen clears circulating parasites by pitting and releases 'once-infected'
RBC deformability varies between
RBCs in circulation. This triggers postartesunate delayed hemolysis and
African subgroups displaying different
explains the long post-treatment positivity of histidine-rich protein 2 (HRP2)- malaria-related phenotypes, pointing to
based dipsticks. Importantly, splenic retention of RBCs also applies to gameto- differences in inherited spleen reactions
cytes, the clearance of which may be enhanced by stiffening them with drugs, to infection.

a potential way to block malaria transmission. The spleen-specific mechanism of

pitting, whereby a poorly deformable
body is extracted from the RBC without
Major Importance of Interactions between RBCs and Spleen in the Pathogenesis lysis, explains the rapid parasite clear-
of Human Malaria ance in artemisinin-treated patients but
Malaria is the most prevalent severe parasitic disease affecting humans. The causal microorgan- also the risk of delayed post-therapeutic
hemolysis.
ism, Plasmodium spp., develops in hepatocytes and then in RBCs, but only the asexual multipli-
cation in RBCs causes clinical manifestations. Invasion of RBCs by Plasmodium triggers major Plasmodium falciparum gametocytes
alterations in this host cell. Plasmodium falciparum, the human-infecting Plasmodium species are initially stiff but become deformable
that has been studied in greatest detail, expresses parasitic proteins at the surface of the RBC upon final maturation which corresponds
to their presence in circulation. Drug-
and modifies its deformability and adhesiveness. Adhesion of parasitized RBCs to endothelial
induced stiffening of mature gameto-
cells (and to other infected or uninfected RBCs) is central in malaria pathogenesis but parasite- cytes is a promising transmission-
induced alterations of RBC deformability have also been associated with the severity of malaria blocking strategy.
[1–3].

White pulp and red pulp structures (see Glossary) have specific functions in the human spleen.
The white pulp is a major operator of the humoral immune response, especially to circulating an-
1
tigens. The red pulp exerts a unique and subtle control of the surface integrity and biomechanical Université de Paris, Biologie Intégrée du
Globule Rouge, UMR_S1134, BIGR,
properties of RBCs. To be left in circulation, RBCs must be fit enough to cross a very specific
INSERM, F-75015 Paris, France
structure of red pulp sinuses, the interendothelial slit (IES; Figure 1). Older RBCs, or RBCs 2
Institut National de la Transfusion
modified by innate or acquired conditions, are eventually retained in the splenic red pulp and Sanguine, 6, rue Alexandre Cabanel,
75015 Paris, France
processed by red pulp macrophages (RPMs) [4]. 3
Laboratoire d’Excellence Gr-Ex, 24,
boulevard du Montparnasse, 75015
The major modifications of RBC biomechanical properties induced by Plasmodium infection point Paris, France
4
APHP, Hôpital Necker Enfants
to the spleen as an important player in the pathogenesis of human malaria. The proportion of sub-
Malades, Service des Maladies
jects with splenomegaly (the splenic index) has long been a defining marker of malaria endemicity. Infectieuses et Tropicales, Centre
Although knowledge has progressed slowly, due to the risk associated with splenic puncture or d'Infectiologie Necker-Pasteur, Institut
Imagine, 149, Rue de Sèvres, 75015
biopsy and limited access to imaging in endemic areas, significant advances have been made on
Paris, France
the role of the spleen in malaria over the last few decades, especially regarding the innate control
of infection and transmission [4,5] (Box 1). Ex vivo perfusion of human spleens [6] and the devel-
opment of biomimetic tools [7–9] have generated new insights. The role of cell-mediated immu- *Correspondence:
nity has been extensively studied in murine models of malaria, and in malaria-infected or exposed pierre.buffet@inserm.fr (P. Buffet).

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© 2020 Published by Elsevier Ltd.
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Figure 1. Interactions between Parasitized and Unparasitized Red Blood Cells (RBCs), Macrophages, and Endothelial Cells in the Splenic Red Pulp,
across Different Physiologic and Pathologic Conditions. (A) Schematic view of the splenic microanatomy. PALS, periarteriolar lymphoid sheath. (B) Representation
of the endothelial lining between splenic cords and venous sinus lumens. Normal RBCs must squeeze through interendothelial slits (IESs) to join venous circulation. Co,
cords of Billroth; SL, sinus lumen. (C) During acute attacks of malaria, splenomegaly can be observed, and very rarely splenic rupture can occur. RBCs infected with

(Figure legend continued at the bottom of the next page.)

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Box 1. Available Tools for Assessing the Human Spleen

Glossary
Spleen Palpation
Cords: circulatory structures specific to
This is the first and the only available assessment in most malaria-endemic settings. Spleen size is best quantified using the splenic red pulp devoid of endothelial
Hackett’s classification. The prevalence of splenomegaly correlates positively with malaria endemicity but lacks specificity. lining. In the cords, circulation of red
Splenomegaly is frequent in children with malarial anemia in endemic areas, but rare in travelers with acute attacks. blood cells is slow, favoring close and
prolonged interactions with splenic
Medical Imaging macrophages which are very abundant
This assesses the size and structure of the spleen and visualizes splenic abnormalities. Ultrasound coupled with Doppler is in this unique environment. CD34+-
the most widely available technique. Splenic contrast-enhanced ultrasonography using microbubbles [94] fine-tunes positive conventional arterioles traverse
splenic evaluation and enables the relative quantification of flow in the fast and slow circulations. Scintigraphy using the red pulp but are not considered to be
radiolabeled tracers provides a functional assessment: sulfur colloids and sensitized RBCs assess macrophage function a component of the slow and open
while heat-stiffened RBCs assess the mechanical filtering function [95]. Splenic function can be inferred from the clearance pathway of the red pulp.
kinetics of labeled RBCs, and from the intensity of radioactivity in the splenic area compared to the hepatic or cardiac area. Ektacytometry: a method for
measuring RBC deformability. Gradually
RBC-Related Markers of Splenic Function increasing shear stress is applied to a
suspension of RBCs diluted in a viscous,
When the filtering and pitting functions are impaired, RBCs containing Howell–Jolly bodies (small Giemsa-positive spheres)
iso-osmolar medium. Measurement of
appear in circulation and can be observed on peripheral blood smears. Pocked RBCs contain small intracytoplasmic ves-
the RBC diffraction pattern enables
icles, visible using differential interference contrast microscopy. Their proportion in the peripheral blood increases above 2–
calculation of the elongation index, a
3% in cases of chronic splenic dysfunction. This accurate marker [96] requires specific equipment and trained personnel.
correlate for RBC deformability.
Erythrophagocytosis: phagocytosis
Circulating Populations of Immune Cells
of generally altered RBCs by effector
These cells, especially IgM memory B cells, have been linked to splenic function. This subpopulation is significantly cells, a process taking place
reduced in splenectomized subjects [97,98]. predominantly in the splenic red pulp
and operated by red pulp macrophages.
Ex Vivo Perfusion of Human Spleens Hyper-reactive malarial
When spleens, retrieved following left pancreatosplenectomy, are perfused ex vivo, their filtering and phagocytic functions splenomegaly (HMS): a rare form of
are preserved for a few hours [6]. This approach has uncovered the innate, mechanical retention of a proportion of ring- chronic malaria, occurring in persistently
infected RBCs [19], a process later confirmed through filtration in vitro [7,20]. exposed subjects, defined by a gross
splenomegaly, elevated total IgM, high
Some biomimetic tools attempt to reproduce splenic function; microsphiltration evaluates the ability of RBCs to squeeze titers of anti-Plasmodium antibody with a
through narrow slits between metallic beads, and accurately reflects mechanical retention of RBCs in the human spleen polyclonal immune response pattern,
[7,8,99]. The initial experimental set-up [7] has been adapted to 96- or 384-well microplates, enabling the parallel analysis and clinical response to antimalarials.
of hundreds of samples [8]. Spleen-mimetic microfluidics chips have been recently developed [9,100]. A stage-dependent Interendothelial slits (IESs): narrow
retention was observed when chips were infused with infected, labeled RBCs. spaces (0.2–2 μm) between adjacent
elongated endothelial cells of sinuses of
the splenic red pulp. Venous sinuses do
not have a basal membrane but a highly
discontinuous helicoid basal fiber. RBCs
must cross interendothelial slits as they
humans through the analysis of circulating cell populations. However, the aforementioned con- navigate from the cords back to the
straints have hampered progress in the understanding of intrasplenic immune responses to ma- general circulation, which challenges
laria in humans. If an important role for platelets in malaria pathogenesis is likely [10], the their deformability. Interendothelial slits
are 2–10 times narrower than the
contribution of intrasplenic platelets, which represent a third of the total human pool, remains
smallest capillaries.
largely unexplored. In addition, substantial anatomic differences exist between human and Pitting: a spleen-specific mechanism
by which an intraerythrocytic body is
extracted from the RBC. The RBC is not
lysed by the pitting process and returns
mature forms are retained in the spleen and processed by red pulp macrophages (RPMs). A proportion of ring-infected RBCs
to the circulation. Pitting plays a major
cross the IESs. Some uninfected RBCs also become stiffer and might also be retained. (D) In the case of malarial infection in a
role in parasite clearance following
splenectomized host, parasitemia is higher, and mature forms, mostly sequestered in peripheral tissues and otherwise
therapy with artemisinins (but not
retained in the spleen of spleen-intact subjects, appear in the peripheral circulation. (E) During chronic parasite carriage,
following treatment with other currently
splenomegaly can be observed inconstantly, and in rare cases it evolves towards hyper-reactive malarial splenomegaly.
available antimalarial agents).
Infected RBCs might be retained like during acute infection. Part of uninfected RBCs may also be retained. An intense
Postartesunate delayed hemolysis:
intrasplenic retention of infected RBCs might lead to a fully intrasplenic replication cycle of the parasite. (F) During acute
acute hemolytic anemia, occurring
malaria attacks treated with artemisinin derivatives, the pitting process occurs in the spleen: dead parasite remnants are
1–2 weeks after treatment with
expelled from the RBC without lysing it. 'Once-infected' RBCs harbor parasite proteins (green dotted line) at the inner side
artemisinin derivatives for severe,
of their membrane, allowing them to be identified in the peripheral blood. In some cases, 1–2 weeks after acute attacks,
generally hyperparasitemic malaria.
synchronous hemolysis of once-infected RBCs is responsible for postartesunate delayed hemolysis (PADH). (G) In
This adverse event is triggered by the
subjects carrying gametocytes, retention of immature forms (stages I to IV) occurs in the spleen due to their reduced
relatively synchronous clearance of
deformability. After evolution towards mature stages (stage V), RBCs infected with gametocytes are able to cross IESs
pitted RBCs.
and therefore circulate in the peripheral blood. Abbreviation: HRP-2, histidine-rich protein 2.

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murine spleens, especially regarding the red pulp which is devoid of bona fide IES in mice [11] Rapid diagnostic tests:
immunochromatographic tests for the
(although mechanical filtration exists), and between human and murine Plasmodium species. diagnosis of Plasmodium parasitemia.
Whether mouse models of malaria are relevant to explore the pathogenesis of human malaria is These tests are performed on capillary
still a matter of controversy [12]. Unlike the spleen of humans and rats, the mouse spleen does blood collected by finger prick and
not have sinuses. The mechanical sensing of RBCs thus depends on holes in venules rather detect the presence of plasmodial
antigens, either panspecific (such as
than on IESs in sinus walls [13]. It is unclear whether these markedly different microanatomic lactate deshydrogenase), or species-
structures display a close or loose functional convergence, and whether the mouse spleen can specific (such as the histidine-rich
pit RBCs containing undeformable bodies, a major treatment-induced parasite clearance pro- protein-2).
cess in humans (please see later: 'Role of the Spleen in Parasite Clearance Following Antimalarial Red pulp: part of splenic parenchyma
involved in the control of RBC
Therapy'). Whether the splenic function is fully preserved in humanized mice infected with biomechanical and surface properties,
P. falciparum is also questionable as stiff immature gametocyte and mature asexual stages – erythrophagocytosis, and reaction to
which should be filtered out by the spleen – are observed in the circulation of these mice [14]. circulating antigens. It includes cords
and venous sinuses.
We have therefore limited this review to observations in humans.
Venous sinuses: circulatory structures
collecting blood downstream from the
Functional Anatomy of the Human Spleen splenic cords and from the closed and
General Features fast perifollicular circulation of the spleen.
The endothelial lining of venous sinuses
The spleen is an oval retroperitoneal organ of 10–12 cm in its major axis, weighing 100–200 g in
is made of parallel, elongated cells lying
adults, surrounded by a collagenous, poorly extensible capsule. It is considered to be poorly con- on discontinuous basal fibers.
tractile in humans compared with diving or fast-running animal species (seals, whales, dogs, White pulp: part of splenic parenchyma
horses) which harbor large 'storage spleens'. Splenic parenchyma is composed of two specific involved in immune responses,
especially against circulating antigens
tissues, the red pulp and the white pulp. The white pulp is made of immune cells, organized in and encapsulated bacteria. It comprises
periarterial lymphoid sheaths containing mostly T cells, and lymphoid follicles containing mostly periarteriolar lymphoid sheaths and
B cells. Follicles are surrounded by the marginal and perifollicular zones. The red pulp accounts lymphoid nodules.
for 75% of splenic volume and is composed of a unique association of cords and venous
sinuses. Cords are circulatory structures that contain fibroblasts and, very predominantly, spe-
cific macrophages (RPMs). Venous sinuses, which collect blood from the cords, are made of en-
dothelial cells, which are parallel to the blood flow and are surrounded by a helicoid basal
fiber (i.e., highly discontinuous) which leaves room for narrow spaces between endothelial cells
(0.2–2 μm), named IESs [13]. Rare plasma cells, mastocytes, and other white blood cells are
also observed in the red pulp.

Microcirculation in the Human Spleen

Overall, about 5% of arterial flow enters the spleen through the splenic artery, and is divided into
two paths: approximately 85% of splenic flow enters a fast, closed circulation, which navigates
around white pulp structures and follows a specific microcirculation from arterioles to venules
delimited by specific MAdCAM1 (Mucosal Addressin Cell Adhesion Molecule 1)-positive cells in
the perifollicular zone; 15% engages into the open and slow circulation in the red pulp, through
the cords, then crosses the unique structure of the IES to join venous sinuses upstream from ve-
nules. Crossing the IES is the most stringent biomechanical challenge for circulating RBCs. RBCs
altered by heat [15], stiffening chemicals [16], genetic defects [17], possibly pretransfusion stor-
age [18], and P. falciparum [19,20] are retained in the cords, where RPMs can process them.

Variable Definitions of the Human Marginal Zone

In rodents, a marginal zone (MZ), containing fibroblasts, dendritic cells, lymphocytes, and specific
macrophages ('metallophilic macrophages' and 'MZ macrophages'), has been described in the
surroundings of lymphoid follicles, and is separated from the white pulp by the marginal sinus
[13]. Such a structure is not present in humans, although an important population of B cells is
found in this area, the MZ B cells, which have a major role in the early, T-independent antibody
response to circulating antigens. Hence, the 'human equivalent of MZ' is sometimes renamed
the perifollicular zone, or as recently proposed, superficial zone [11].

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Malaria in Splenectomized Subjects: A More Severe Disease?

From 1931 through 2019, 133 malaria attacks were reported in splenectomized patients, among
which 69 were due to P. falciparum, 26 to Plasmodium vivax, six to Plasmodium knowlesi, and
three to Plasmodium malariae. Infection was mixed in one case, and in 28 cases the Plasmodium
species was not reported [4] (see Table S1 in the supplemental information online). Only two co-
horts assessing the risk of malaria in splenectomized subjects (33 in Malawi, 11 in Indonesia) have
been prospectively followed and compared with controls. In both cohorts, acute malaria attacks
were significantly more frequent in splenectomized patients [21,22]. In this population, severe at-
tacks are also more frequent, mature forms of P. falciparum – which are normally sequestered by
adherence to the endothelium – are observed in circulation, and post-treatment parasite clear-
ance is delayed [23,24]. However, the overincidence of severe forms and mortality has not
been confirmed by prospective controlled studies and its high frequency in published case re-
ports or series may either reflect a genuine oversusceptibility or result from a publication bias.
An increased risk of acute P. vivax attacks after splenectomy has been recently reported, and out-
weighs the risk of P. falciparum attacks (hazard ratio 7.7 vs 2.3) [22], without evidence for an in-
creased risk of severe forms in this immune population. One case of acute malaria due to
P. knowlesi has been described in an asplenic patient [25], and in another case a prolonged par-
asite clearance has been reported [26]. In summary, the increased risk of malaria has been ro-
bustly described in immune or semi-immune subjects, and is strongly suspected in
nonimmune travelers [27]. Nonimmune splenectomized hosts should be very cautious when trav-
eling to malaria-endemic areas. If these patients must travel to endemic areas, strict compliance
to antimalarial prophylactic measures is warranted.

The Spleen in Acute Malaria

Protective Role
The human spleen senses subtle alterations in RBC deformability. This decreased deformability of
Plasmodium-infected RBCs (asexual stages), first observed with P. knowlesi [3], then with
P. falciparum [1], is intense in RBCs infected by mature forms, but is already present, albeit mod-
erately, at the ring stage [1,7,19,20,28]. Mechanisms include alterations of surface-to-volume
ratio [20], and interactions between parasite proteins and the RBC cytoskeleton [29,30] which in-
duce membrane rigidification. Importantly, ektacytometry – a reference technique to quantify
RBC deformability [31] – and microfluidics have also demonstrated that, during acute malaria at-
tacks, even noninfected RBCs are less deformable than normal control RBCs [2,28,32].

As evidenced by ex vivo perfusions of human spleens with P. falciparum-infected RBCs, the

spleen retains a proportion (approximatively half) of ring-infected RBCs, in addition to the ex-
pected, almost complete, retention of schizont-infected RBCs [19]. The mechanical retention of
schizont-infected RBCs is expected to display a small pathogenic impact in vivo, as the majority
of mature forms cytoadhere in other organs, which prevents them from being filtered by the
spleen. Retention of rings, which affects a predominantly circulating component of the parasite
biomass, takes place in the red pulp. This mechanism, which is difficult to explore directly in
human subjects, is expected to reduce the circulating parasitic load, thereby also reducing the
parasite biomass amenable to sequester in other organs [19].

The increased clearance of all RBCs (infected and uninfected) by the spleen after acute malaria
has been demonstrated through the autotransfusion of radiolabeled RBCs. Thai patients with
acute malaria had indeed an enhanced clearance of heat-stiffened RBCs when splenomegaly
was present [15] that also affected labeled normal RBCs in a similar postmalarial context [33].
This enhanced splenic clearance of abnormal and normal RBCs, which lasts for several weeks,
may contribute to the partial control of parasite loads. When comparing cerebral malaria with

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other severe forms (anemia, respiratory distress, acute renal and/or liver failure, shock), postmor-
tem studies also showed that intrasplenic erythrophagocytosis was more frequent in the latter
[34]. Splenomegaly is also less frequent in cerebral malaria than in severe malarial anemia, as
demonstrated by field studies performed in Uganda and Sudan, and including a total of 1108 chil-
dren [35–37]. In one of these studies, splenomegaly was also associated with a survival advan-
tage [37]. The human spleen can thus retain part of the parasite biomass, which may partially
control the course of infection, potentially reducing the risk of cerebral malaria. This would, how-
ever, come at the expense of an increased retention and clearance of noninfected RBCs, leading
to splenomegaly and anemia (Figure 1). Exploration of the innate retention of (dead or live) rings in
the controlled human infection model [38] would be the most direct and relevant way to assess
whether this potentially powerful protective process indeed exists, in a context of limited splenic
activation at early stages of human infection, when splenomegaly is not yet present [15].

Detrimental Role
The pathogenesis of acute malarial anemia is complex and only partially understood. It is associ-
ated with intra- and extravascular hemolysis, dyserythropoiesis, and the clearance of larger pro-
portions of uninfected than infected RBCs [39]. Several observations point to a splenic
contribution to the pathogenesis of acute malarial anemia. Congestion of the red pulp is a key
postmortem feature in fatal malaria [40,41]. Decreased RBC deformability, which triggers
intrasplenic retention ex vivo (see above), correlates with a hemoglobin nadir [32]. As stated
above, splenomegaly is more prevalent in patients with severe malarial anemia than in other se-
vere forms, and splenic clearance of RBCs is increased after malarial attacks. Not least,
erythrophagocytosis is enhanced in acute malaria, and alterations of infected RBCs render
them prone to phagocytosis [42]. The phagocytic function of monocytes is also activated in ma-
laria [43]. That erythrophagocytosis in malaria predominantly takes place in the macrophage-rich
spleen remains to be directly demonstrated in humans.

Very rarely, splenic-specific complications of malarial infection occur: pathologic splenic rupture,
intrasplenic hematoma, and splenic infarction. These complications have received little attention
so far, and their precise mechanisms remain elusive [44]. In summary, multiple observations sug-
gest a protective role for the spleen in acute malaria, even if definitive evidence from controlled stud-
ies is lacking. The innate retention of rings can prevent the rise of peripheral parasite load thereby
decreasing the risk of cytoadherence and sequestration of parasitized RBCs in vital organs [45].
The potential downside of this phenomenon is a splenic contribution to malarial anemia.

Role of the Spleen in Parasite Clearance Following Antimalarial Therapy

The Pitting Process
In patients recently cured of malaria by artemisinin derivatives, a proportion of circulating RBCs
display parasite proteins at the internal layer of their plasma membrane [46]. These 'once-
infected' RBCs do not contain parasites [23,46,47] and their presence results from a spleen-
specific physiological process called pitting, whereby an intraerythrocytic body is extracted
from the RBC without cell lysis [48]. Pitting clears physiological intraerythrocytic items such as
Howell–Jolly or Pappenheimer bodies. Malaria-related pitting can be quantified in the peripheral
blood of patients through the double-staining of RBCs with a nucleic acid marker and an antibody
against parasite proteins such as RESA (ring-infected erythrocyte surface antigen). Pitted RBCs
are parasite-negative and parasite-membrane-protein-positive. Pitting is not observed in
splenectomized subjects [23], and has been replicated in human spleens perfused ex vivo with
parasitized RBCs pre-exposed to artemisinin derivatives [6]. Pitting does not occur (or occurs
to a very low level) with RBCs containing live parasites, and so far has been explored only in
P. falciparum infections.

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Contribution of Pitting to Parasite Clearance

In nonimmune travelers treated with artemisinins, pitting is a major mechanism of parasite clear-
ance [49]. In artemisinin-treated children living in malaria-endemic Mali, clearance mechanisms
may be more complex and influenced by the immune status of the host. Pitting appears to be
an important contributor to clearance in infants, who are expected to have low antiparasite
humoral immunity; however, in older children, immunity correlates with an even faster parasite
clearance, likely through immune-mediated phagocytosis, and possibly through splenic retention
without pitting [50]. Pitting is marked in patients treated with intravenous artesunate or oral
artemisinin derivatives, and much lower after treatment with quinine, mefloquine, or
atovaquone-proguanil [51].

Contribution of Pitting to Postartesunate Hemolysis

In Japanese, then in German, travelers treated with artesunate for hyperparasitemic severe ma-
laria [52,53], acute hemolysis was observed 1–2 weeks after therapy. These hemolytic episodes
were moderately intense and affected 5–27% of travelers [54,55]. Transfusion was required in 5–
50% of cases [56] and no conventional cause of hemolysis was found. Initial observations in
Southeast Asia had shown that once-infected RBCs have a shorter lifespan than normal RBCs
[47]. In travelers, these hemolytic episodes (named postartesunate delayed hemolysis,
PADH) and the disappearance of once-infected RBCs from the circulation were simultaneous.
PADH is thus likely triggered by the clearance of once-infected RBCs [57]. The peak concentra-
tion of once-infected RBCs, quantified by flow cytometry, 2–7 days after treatment initiation, is
predictive of PADH [57,58]. Predicting PADH in resource-limited settings is important, as weekly
follow-up of all patients recovering from severe malaria and preparation of transfusion are logisti-
cally demanding. Like RESA, the parasitic histidine-rich protein 2 (HRP-2) is left as an imprint at
the membrane of pitted RBCs [58]. HRP-2 is the antigen used in many immunochromatographic
malaria rapid diagnostic tests. The persistent positivity of the diagnostic dipstick, performed on
diluted whole blood 3–7 days after initiation of artesunate, accurately predicted PADH [58].
Whether PADH is a significant problem in endemic areas has been a subject of debate; a pro-
spective study following 217 Congolese children with acute malaria, treated with quinine or intra-
venous (IV) artesunate, and followed until 42 days after treatment, showed PADH in less than 1%
of cases [59]. These patients, however, had parasitemia below the conventional 10% threshold
for hyperparasitemia and had uncomplicated malaria. Loosely defined delayed hemolysis has
been observed in 7% of African children treated with artesunate for severe malaria [60]. Further
prospective studies in African children are ongoing. Interestingly, this approach also explains
why HRP-2-based dipsticks remain positive days to weeks after treatment of a malaria attack
with artemisinins. HRP-2 persists in the circulation not as soluble protein in plasma but as a
cytoskeleton-associated protein in once-infected RBCs.

The Spleen in Chronic Parasite Carriage

A Splenic Contribution to Chronic Malarial Anemia?
The mechanisms of malarial anemia are influenced by the infecting Plasmodium species, age, and
transmission intensity, amongst other factors. Excellent reviews have dissected these complex
parameters [39,61]. Briefly, most important contributors are intra- and extravascular hemolysis,
erythrophagocytosis, dyserythropoiesis (favored by frequent comorbidities such as iron or vita-
min B12 deficiency, or malnutrition), some degree of bone marrow insufficiency, and possibly
splenic clearance. The role of the spleen in chronic parasite carriage is complex and very difficult
to explore. One study, carried out in Ghana [62], found a relationship between chronic parasite
carriage, anemia, and splenomegaly, as observed in multiple other populations and settings.
This may reflect any combination of long-lasting RBC trapping in the spleen, enhanced
erythrophagocytosis or increased immune reactivity.

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Parasite Biomass in the Spleen

Recently, comprehensive pathological and molecular examinations of spleens (and concomi-
tantly of peripheral blood) in mostly untreated patients undergoing splenectomy in a highly
P. falciparum- and P. vivax-endemic area of Indonesia have shown that, compared with periph-
eral blood, nonphagocytosed parasitized RBCs concentrate in the spleen [63]. These observa-
tions not only bring some substance to the protective role of the spleen filter in parasite
clearance but also suggest that the spleen may be a parasite shelter, possibly supporting a cryp-
tic intrasplenic parasitic cycle [4,64].

Hyper-Reactive Malarial Splenomegaly

This condition, initially named 'tropical splenomegaly syndrome', then more precisely de-
scribed in 1957, has been progressively deciphered over the last decades, the name
hyper-reactive malarial splenomegaly (HMS) being proposed in 1983 [65]. The currently
accepted diagnostic criteria for HMS associate marked splenomegaly, total plasma IgM
above two times the local standard deviation, clinical response after antimalarials, and poly-
clonal lymphocytic response [66]. HMS is believed to account for an important proportion
(40%) of massive splenomegaly in malaria-endemic areas [67], but recent and precise epi-
demiological data regarding its prevalence in the general population are scarce. In the
Gambia, three decades ago, the prevalence of HMS was estimated at 0.16% [68]. HMS
has essentially been described in subjects chronically exposed to the parasite and is very
rare in travelers. Typically, patients present with a gross and constant splenomegaly, some-
times clinically patent (abdominal discomfort) and inconstantly associated with hepatomeg-
aly. Total IgM are markedly elevated, with a polyclonal pattern. This elevation usually
precedes splenomegaly. Cytopenia of variable importance can be present, usually attributed
to hypersplenism. Positive diagnosis of malaria is difficult in HMS, as parasite load is usually
very low, and only detected by molecular methods. Biological features of autoimmunity
(cryoglobulinemia, rheumatoid factor, antinuclear antibodies) have been associated with
HMS. Recently, a relatively specific pattern of antinuclear antibodies has been associated
with chronic malaria [69].

A genetic component of HMS is suspected, based on familial studies [70], but its precise basis
remains unknown (although it is certainly not Mendelian). Pathologic examination of the spleen
in HMS has been rarely reported; published cases described essentially red pulp congestion
and erythrophagocytosis, with only brief descriptions of the white pulp [71–73]. The pathogen-
esis of HMS is poorly understood, the most commonly accepted hypothesis involving a defect
in regulatory T cells, leading to a B cell expansion after malaria infection, production of immune
complexes which would be eventually phagocytosed by splenic macrophages. If this hypoth-
esis is in line with splenomegaly and the enhanced immune response observed in HMS, it how-
ever falls short in providing an explanation for anemia and lower parasitic load. The evolution of
HMS in endemic areas remains largely unknown. Historical data pointing to a very important
mortality (N50%) might be biased by the imprecision regarding the causes of death [74]. Scarce
data have been collected in travelers, showing slow regression of hyper IgM and splenomegaly
over several months [75]. Travelers or expatriates affected by HMS exhibit a tendency to suffer
from similar episodes of HMS upon re-exposure to the parasite, consistent with an innate path-
ogenic component and genetic susceptibility [76]. A major issue is the risk of evolution towards
a low-grade marginal-zone splenic lymphoma (MZSL). This association, suspected 50 years
ago, has been essentially observed in West Africa. MZSL shares many features with HMS. In
one study, B cell receptor clonality was assessed in Ghanaian patients with HMS separated ac-
cording to their response to antimalarials: clonality was absent in responders, constant in non-
responders, and observed in two out of 13 partial responders [77]. These findings, along with

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Trends in Parasitology

others, led to the hypothesis that splenic lymphoma represents an ultimate evolution of un- Outstanding Questions
treated HMS [78], but this risk has not been precisely quantified. Epidemiology, physiology of malaria,
and alterations of splenic function – is
the risk of malaria higher in nonimmune
Antimalarial treatment of HMS relied on prolonged courses of primaquine, proguanil, mefloquine,
splenectomized individuals or in sub-
or chloroquine. In travelers not returning to malaria-endemic areas, a short regimen is as effective jects with partial impairment of splenic
as prolonged treatments [76]. The value of such short courses remains unknown in endemic function? In splenectomized subjects,
areas and the usual policy is to administer an intermittent prophylaxis to HMS patients persistently do other macrophage-rich organs,
such as the liver or the lung, operate
exposed to the parasite [79]. Splenectomy may prove dangerous in HMS patients as it carries a
antiplasmodial defense as efficiently
risk of postoperative acute malaria [64,80,81] and exposes the patient to the long-term risks as- as the spleen?
sociated with splenectomy [82].
Role of the spleen in acute attacks –
The Fulani Enigma are the ring-infected RBCs retained in
the spleen intrinsically different from
Fulani subjects, present across Sahelian Africa (from Southern Sudan to Eastern Senegal), used those flowing through? Is the differ-
to be nomad pastoralists. In Mali and Burkina Faso, compared with other sympatric subjects, ence related to the parasite or to the
Fulani exposed to Plasmodium display a distinguishable phenotype, with frequent splenomegaly, host RBC? Which parasitic proteins
and host receptors are particularly in-
enhanced antiplasmodial humoral response, lower body temperature, lower hemoglobin, and
volved in the splenic retention of in-
lower parasitic load. For these reasons, Fulani have been considered as protected from malaria. fected RBCs? To what extent does
This phenotype is, however, neither complete nor constant (Table S2), the most constant feature intrasplenic erythrophagocytosis con-
being palpable splenomegaly. The determinants of the 'Fulani phenotype' have been mostly tribute to severe malarial anemia?
studied from an immunological standpoint, showing, in Fulani, an activated state of monocytes Pathogenic role of the spleen in
[83], enhanced proinflammatory cytokine production [84], a strong response of dendritic cells chronic infection – what are the relative
to Toll-like receptor agonists [85], and a defect in regulatory T cells [86]. The genetic basis of contributions of intrasplenic mechani-
this phenotype remains unclear as the conventional RBC polymorphisms associated with malaria cal retention and phagocytosis of
RBCs in chronic malarial anemia?
protection have not been found to be more frequent in the Fulani [87]. The Fulani phenotype is, in
many aspects, reminiscent of HMS, and HMS is over-prevalent in the Fulani [68]. Comprehensive, Immune function of the human spleen
multidisciplinary field studies are underway to decipher the determinants of this phenotype and to in malaria – what is the contribution of
MZ B cells in the early humoral immune
deconvolute the relative contributions of RBC-spleen interactions and genetics [88].
response to Plasmodium, especially
the increasingly recognized IgM
Role of the Spleen in the Transmission of P. falciparum response?
Gametocytes, initial sexual forms of Plasmodium in RBCs, are generated in a small proportion
Role of the intrasplenic platelets pool in
(0.1–5%) at each asexual replication stage. They progressively mature from stage I to stage IV malaria – is there a role for platelet-
(immature gametocytes) to stage V (mature forms) over 14 days, under the influence of external mediated parasite killing inside the
factors (reviewed in [89]) and the transcription factor PfAP2-G. The deformability of gametocytes spleen, and could this component par-
ticipate in the modulation of circulating
is markedly reduced from stages I to IV, then it drastically improves [90]. Immature stages accu- parasite biomass?
mulate in the spleen and in extravascular spaces of the bone marrow [91,92] and are generally
absent from the peripheral circulation, unlike mature stages. Circulation of mature gametocytes
is a prerequisite for the parasite to be ingested by Anopheles and to maintain the parasite's trans-
mission cycle. The deformability of immature gametocytes is regulated in part by the parasite pro-
tein STEVOR, kinase A, and AMPc [93]. A pharmacological approach aiming at artificially
stiffening mature gametocytes has been developed with the aim of inducing their mechanical re-
tention in the spleen, which would subsequently prevent their circulation and make them unavail-
able to Anopheles, hence blocking transmission. This approach has benefitted from the
technological improvements of microsphiltration (Box 1). A large, high-throughput screening
campaign to discover stiffening compounds is ongoing [8].

Concluding Remarks
The spleen has been extensively investigated, in animal models and in humans, from an immuno-
logical perspective. The exploration of how the spleen innately filters normal and altered RBCs,
and eventually eliminates them (predominantly in the red pulp), had received attention from re-
searchers half a century ago. New tools have enabled progress in this field over the last decade.

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Key Table
Table 1. Dual Role of the Spleen in Human Malaria
Acute attacks In treated patients Chronic carriage Transmission
Protective Retention of infected Clearance of infected RBCs Limitation of the circulating
Retention of gametocytes
role RBCs (pitting) parasitic load?
- Contribution to chronic anemia?
- Rupture
Detrimental Postartesunate delayed - Hyper-reactive malarial Reservoir? (contribution to chronic
- Contribution to
role hemolysis splenomegaly asymptomatic carriage)
anemia
- Splenic lymphoma?

A better understanding of human splenic physiology has shed light on the dual role 'Dr Jekyll and
Mr Hyde-like' of the spleen in human malaria (Table 1, Key Table). The spleen limits the increase in
parasite biomass, therefore reducing sequestration and microvascular dysfunction in major target
organs like the brain, but concomitantly likely contributes to malarial anemia. Similarly, the spleen-
related production of once-infected RBCs after treatment with artemisinins reduces RBC loss
during therapy but sometimes induces delayed and clinically significant hemolysis. Current and
further lines of research (see Outstanding Questions) will investigate the promising field of induc-
tion of splenic retention of Plasmodium gametocytes, a potentially important contributor to ma-
laria elimination attempts. Not least, new exploratory tools (Box 1) will be of importance to
investigate the role of cellular immunity antimalarial defense, a process essentially studied through
in vitro studies and animal models so far.

Acknowledgments
The authors wish to thank Dr Geneviève Milon for her longstanding support and pertinent scientific guidance; Dr Steven Kho
and Professor Nicholas Anstey for their comments on this manuscript and fruitful collaboration.

Supplemental Information
Supplemental information associated with this article can be found online at https://doi.org/10.1016/j.pt.2020.03.001.

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