Trends in

Parasitology
Review

The biology and pathogenesis of vivax malaria

Nicholas M. Anstey 1,*, Wai-Hong Tham 2,3,4, G. Dennis Shanks 5,
Jeanne R. Poespoprodjo 1,6,7,8, Bruce M. Russell 9, and Steven Kho 1,7

Plasmodium vivax contributes significantly to global malaria morbidity. Key ad- Highlights
vances include the discovery of pathways facilitating invasion by P. vivax merozo- Most asexual vivax parasites accumulate
ites of nascent reticulocytes, crucial for vaccine development. Humanized mouse in the reticulocyte-rich spleen, replicating
in an endosplenic life cycle. This reservoir
models and hepatocyte culture systems have enhanced understanding of
contributes to systemic inflammation
hypnozoite biology. The spleen has emerged as a major reservoir for asexual and anemia, and helps maintain chronic
vivax parasites, replicating in an endosplenic life cycle, and contributing to recur- infections and transmission.
rent and chronic infections, systemic inflammation, and anemia. Splenic accumu-
Plasmodium vivax merozoites preferen-
lation of uninfected red cells is the predominant cause of anemia. Recurring and tially invade nascent reticulocytes over
chronic infections cause progressive anemia, malnutrition, and death in young mature reticulocytes through newly iden-
children in high-transmission regions. Endothelial activation likely contributes to tified invasion pathways.
vivax-associated organ dysfunction. The many recent advances in vivax pathobi-
Humanized mice and hepatocyte culture
ology should help guide new approaches to prevention and management. systems are characterizing hypnozoite
biology, and new approaches for anti-
relapse therapy.

Why vivax malaria matters Anemia is mostly from splenic retention

P. vivax remains a major cause of morbidity in malaria-endemic regions, having caused an esti- of uninfected red cells; frequently recur-
ring and chronic infections cause pro-
mated 6.9 million clinical cases of malaria in 2022 [1], and many more chronic asymptomatic infec-
gressive anemia, malnutrition, and
tions [2–4]. It is capable of causing severe and fatal malaria, including progressive severe anemia death, especially in young children.
and malnutrition, arising from its propensity to cause relapsing and chronic infections [3,5–7],
and hypotension and organ dysfunction in acute infections [8–10]. However, understanding of un- Endothelial cell activation, a predictor
of impaired perfusion and death in
derlying mechanisms and the role of comorbidities remains incomplete. Relapsing infections and
falciparum malaria, is even more pro-
chronic low-density infections also contribute to transmission [5], making control and elimination nounced in acute vivax malaria, likely
of vivax malaria more difficult than falciparum malaria. Much has been learnt about the P. vivax contributing to organ dysfunction in se-
life cycle and its biology and pathogenesis in recent years that is important in advancing the preven- vere disease.

tion, management, and elimination of this major pathogen. 1

Global and Tropical Health Division,
Menzies School of Health Research,
The P. vivax life cycle: predominance of extravascular replication Charles Darwin University, Darwin,
Recent findings have fundamentally changed our understanding of the P. vivax life cycle Northern Territory, Australia
2
Walter and Eliza Hall Institute of Medical
(Figure 1, Key figure). While relapse (see Glossary) from latent hypnozoites [11] remains a car- Research, Parkville, Victoria, Australia
3
dinal feature of P. vivax biology, it is our understanding of the asexual-stage life cycle, and in Department of Medical Biology,
particular the identification of a hitherto unrecognized endosplenic life cycle, that has been University of Melbourne, Melbourne,
Victoria, Australia
transformed [3]. 4
Research School of Biology, Australian
National University, Canberra, ACT,
Asexual replication in the reticulocyte-rich spleen Australia
5
School of Public Health, University of
Recent studies show that P. vivax asexual stages are not confined to the peripheral circulation as Queensland, Brisbane, Queensland,
long thought. Indeed, vivax malaria is predominantly an infection of the spleen, with only a small Australia
6
circulating intravascular component [12]. In chronic infection, over 98% of total-body P. vivax par- Centre for Child Health and Department
of Child Health, Faculty of Medicine,
asites are found in the spleen [12], sustained by an endosplenic asexual-stage life cycle [12,13] Public Health and Nursing, Universitas
(Figure 1). Multiple lines of evidence support the existence of a major cycle of intrasplenic replica- Gadjah Mada, Yogyakarta, Indonesia
7
tion. The magnitude of asexual-stage P. vivax biomass accumulating in the spleen is inexplicable Timika Malaria Research Facility,
Papuan Health and Community
by retention of peripherally produced parasites alone, even after incorporating highly conservative Development Foundation, Timika,
estimates against an endosplenic life cycle [12,13]. The spleen contains all asexual developmental Central Papua, Indonesia

Trends in Parasitology, July 2024, Vol. 40, No. 7 https://doi.org/10.1016/j.pt.2024.04.015 573

Crown Copyright © 2024 Published by Elsevier Ltd. All rights reserved.
 Trends in Parasitology

8
Key figure Mimika District Hospital and District
Health Authority, Timika, Central Papua,
The Plasmodium vivax life cycle Indonesia
9
Department of Microbiology and
Immunology, University of Otago,
Dunedin, New Zealand
Plasmodium vivax Mosquito
cycle ookinete
oocyst
life cycle gametes
zygote *Correspondence:
nicholas.anstey@menzies.edu.au
(N.M. Anstey).

sporozoites Splenic reservoir and

endosplenic cycle

Pre-erythrocytic erythroblastic
liver phase island

hepatocyte

hypnozoite
merosome

merozoites ?
ring
erythroblast

uninfected
reticulocyte

trophozoite
gametocyte
schizont

Circulating Bone
erythrocytic cycle marrow niche

Trends in Parasitology

Figure 1. Sporozoites from female anopheline mosquitos enter the human host following a blood meal and travel to invade
liver hepatocytes. Pre-erythrocytic incubation of 1–2 weeks results in release of merozoites from hepatic schizonts and
invasion of circulating nascent reticulocytes, commencing a 48 h intraerythrocytic asexual life cycle that is coupled with the
human circadian rhythm [179]. Within reticulocytes, asexual parasites develop from ring stages to trophozoites and
multinucleated schizonts which rupture to release merozoites that reinvade other reticulocytes, in both the intravascular
and extravascular (spleen and bone marrow) compartments, initiating a new asexual cycle. An endosplenic life cycle
sustains a large extravascular biomass of asexual P. vivax in the spleen. A fundamental feature of the P. vivax life cycle is
the dormant hypnozoite stage within hepatocytes, which can activate weeks or months after the initial infection, giving rise
to recurrent blood stages and relapse. Release of asexual parasites from the spleen back into the circulation, and relapses
from hepatic hypnozoites, sustain a high prevalence of chronic low-density, often asymptomatic, infections. Sexual
commitment occurs during merozoite development in intraerythrocytic schizonts or earlier in the liver phase. Gametocyte
development is rapid (2–3 days) and occurs concurrently with asexual stages, predominantly in circulation and the bone
marrow. Infectious gametocytes are ingested by a competent Anopheles mosquito, in which they undergo sexual followed
by asexual reproduction to form thousands of sporozoites for onward transmission. This figure was created using BioRender.

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stages of P. vivax, and their proportions are consistent with their durations in its 48 h life cycle (re- Glossary
viewed in [13]). CD71-expressing nascent reticulocytes, the preferred invasion targets for 8-aminoquinolines: drugs including
P. vivax [14,15], are enriched in the splenic red pulp and colocalize with more than 90% of asexual primaquine and tafenoquine that kill
latent hypnozoites and asexual stages of
P. vivax parasites [13]. The physiological properties of the slow circulation in the red-pulp cords, in P. vivax.
combination with the observed magnitude and localization of P. vivax and its target cells, offer Acute kidney injury (AKI): a
ideal conditions for successful invasion–reinvasion events [13]. complication reported in severe vivax
malaria.
Acute respiratory distress
Nascent reticulocytes released by the bone marrow have been recently found to be physiologi- syndrome (ARDS): a pulmonary
cally retained in the spleen for final maturation [13], and likely accumulate through multiple mech- complication seen in severe vivax
anisms. The rigid nature of nascent CD71+ reticulocytes suggests biomechanical retention in the malaria.
CD71: also known as transferrin
cords upstream of inter-endothelial slits [13,16], compounded by increased splenic filtration strin-
receptor 1 (TfR1), a receptor
gency in chronic malaria [17]. CD71+ reticulocytes are also observed lining sinus lumen walls, predominantly present on nascent
suggesting that ligand-based interactions and cytoadherence to endothelial cells facilitate reten- reticulocytes and absent on mature
tion in this region [13,16,18]. The greater proportion of adherent reticulocytes in vivax- compared ones, playing a key role in invasion by
P. vivax merozoites.
to falciparum-infected spleens [13] suggests that reticulocyte cytoadherence may also be differ-
CD98: also known as 4F2 cell-surface
entially regulated in P. vivax infections to enhance cytoadherence of its target cell [18]. Erythro- antigen heavy chain or CD98hc, a
blastic islands, a marker of erythropoiesis not physiologically seen in the human spleen and multifunctional transmembrane protein
only rarely seen in human disease, have recently been observed in P. vivax-infected spleens with a key role in reticulocyte invasion by
P. vivax merozoites. Expressed in
from a high-transmission area, suggesting a remarkable induction of intrasplenic production as various cell types, such as erythrocyte
an additional source of nascent reticulocytes in chronic vivax infection [19]. progenitors and nascent reticulocytes.
Complement receptor 1 (CR1): a
Asexual stages in the bone marrow complement regulatory protein on the
RBC surface and an RBC receptor for
The bone marrow is another primary site for accumulation of P. vivax asexual stages [20–22] P. vivax erythrocyte binding protein
(Figure 1). Parasite densities approximate those in peripheral blood [20,21], though parasites can (PvEBP).
sometimes be found in individuals whose peripheral blood is microscopy-negative [23]. In asymp- Duffy antigen receptor for
chemokines (DARC): a reticulocyte
tomatic infections, the relative contribution of bone marrow parasites to total-body P. vivax asexual
receptor for Duffy-binding protein (DBP),
biomass has been estimated to be 0.1% [13]. Direct comparison of matched human blood, spleen, that mediates merozoite invasion of
and bone marrow in natural human infections is needed to confirm this and to fully understand the reticulocytes.
role of each of these compartments. Histological analysis is mostly only available for non-human Endosplenic life cycle: the cycle of
intraerythrocytic asexual stages that
primate models with the spleen removed [24,25]. Comparison with a spleen-intact non-human pri-
occurs within the spleen.
mate suggests that, in the absence of a spleen, P. vivax accumulation shifts markedly from the Nascent reticulocytes: immature red
spleen to the liver, lung, and bone marrow [25]. Notwithstanding this limitation of splenectomized blood cells (RBCs) characterized by their
models, of the parasites accumulating in the bone marrow, most early asexual stages are retained expression of CD71 (transferrin receptor
1, TfR1+) and positivity for thiazole
in the extravascular hemopoietic spaces, while schizonts are found largely in the vascular sinusoids
orange, signifying the presence of
[24]. This distribution pattern supports the hypothesis that entry into extravascular spaces through reticular matter. Primary target cells for
tight sinusoidal channels may be limited to merozoites [14], and invasion of the large extravascular P. vivax merozoite invasion. Key
pool of erythroid precursors, including erythroblasts [23,26], reflects the high frequency of early receptors involved in this invasion
process, CD71 (TfR1) and CD98, are
asexual stages confined in this region. Human histological data reflecting the specific architectural found on nascent reticulocytes but not
distribution of bone marrow P. vivax are lacking but needed. Bone marrow dyserythropoiesis, on mature class IV reticulocytes, which
widely reported in vivax malaria [20,21], may contribute to changes in the stringency/leakage of are thiazole orange positive but CD71
bone marrow cellular transit channels, potentially providing pathways for increased trafficking of negative.
P. vivax Duffy binding protein
parasitized cells out into the circulation. (PvDBP): a protein that binds to DARC
and is a leading vaccine candidate.
Sexual stages and transmission P. vivax reticulocyte binding protein
2a (PvRBP2a): a protein that binds to
The key biological properties of P. vivax that enhance its transmission have been reviewed else-
CD98 on nascent reticulocytes,
where [4,27,28]. Humanized mouse models offer the potential to further understand the biology mediating invasion.
of both sexual and asexual stages [26]. Gametocyte maturation requires only 2–3 days, much P. vivax reticulocyte binding protein
faster than Plasmodium falciparum, with sexual stages appearing at the same time as asexual 2b (PvRBP2b): a protein that binds to
CD71 (TfR1) on nascent reticulocytes,
stages, allowing transmission prior to the onset of symptoms [3,27]. The capacity of P. vivax in-
mediating invasion.
fections to relapse results in multiple recurrences of concurrent asexual- and sexual-stage

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parasitemia that sustains transmission over time. P. vivax is also capable of maintaining a high Relapse: recurrent P. vivax parasitemia
arising from activation of dormant (latent)
prevalence of asymptomatic low-density mixed asexual and sexual-stage infections [29]. While
hypnozoites.
transmissibility is proportional to gametocyte density [27,30,31], low-density, even subpatent Rosette: a cellular formation in which an
(microscopy-negative) gametocyte infections can result in transmission [31]. The high prevalence infected red blood cell (iRBC), typically
of low-density sexual-stage carriage in asymptomatic infections gives rise to a significant propor- containing a malaria parasite, adheres to
and clusters with multiple uninfected red
tion of transmission in endemic areas [29]. Finally, P. vivax can infect a greater variety of Anoph- blood cells (uRBCs).
eles mosquitoes than P. falciparum and has an ability to develop in the mosquito at lower Subpatent infections:
temperatures, enabling a wider geographic distribution than that of P. falciparum [4]. submicroscopic infections in peripheral
blood, infections below the detection
limit of microscopy.
Human volunteer studies suggest that P. vivax transmission can occur as soon as 7 days after Transferrin receptor 1 (TfR1): also
sporozoite inoculation before the onset of symptoms [32]. This is supported by transcriptomic referred to as CD71, it specifically binds
signatures of early gametogenesis in both hepatic-stage parasites [33] and the first blood- and internalizes transferrin. It is
particularly relevant to the mechanisms
stage cycle following liver-stage development [34], suggesting sexual commitment in primary in-
of merozoite invasion as it relates directly
fection can take place during liver-stage development [33,34] and in response to host physiolog- to the protein's specific function in this
ical signals [33]. Sexual stages derive from circulating asexual-stage parasites [27,35], with rapid process.
maturation times and with all P. vivax gametocyte stages found in circulation [27,35]. Gametocyte VIR: P. vivax proteins expressed by the
large subtelomeric multigene family, vir.
density is highly correlated with asexual parasite density [27,30]. Chronic asymptomatic P. vivax
von Willebrand factor (vWF): a blood
infections are highly prevalent in vivax-endemic areas and persist for much longer than glycoprotein promoting hemostasis,
P. falciparum infections [2]. Longitudinal oscillations of parasite density result in intermittent especially platelet adhesion.
higher-density infections having greater propensity for transmission [2]. Nevertheless, with the
declining incidence of clinical disease, an increasing proportion of the infectious reservoir is com-
prised of low-density asymptomatic infections [28,36]. In the Amazon, subpatent and asymptom-
atic (patent or subpatent) infections are estimated to account for up to 25% and 79% of
community P. vivax transmission, respectively [29].

In addition to peripheral blood, P. vivax gametocytes also develop and mature in the bone marrow
[20,21]. These human studies are supported by splenectomized non-human primate models in
which a large number of immature P. vivax gametocytes are seen in the bone marrow paren-
chyma [24]. Cytoadherence of P. vivax gametocytes to bone marrow endothelial cells may ex-
plain bone marrow accumulation [37]. Gametocytes may also be found in the spleen in acute
illness [38]. In chronic infection, splenic gametocytes appear to be less frequent [13]. It has
been speculated that schizonts accumulating in the splenic perifollicular zones release sexually
committed merozoites that naturally flow through the fast circulation, and may invade uninfected
reticulocytes cytoadhering further downstream in sinus lumina, enabling gametocytes to exit
through the splenic vein for maturation and transmission [13].

Pathobiology
P. vivax reticulocyte invasion
P. vivax pathobiology is intricately linked to the parasite's erythrocytic development cycle. P. vivax
preferentially invades nascent reticulocytes [14] and uses at least three different human red blood
cell (RBC) receptors for parasite invasion (Figure 2): Duffy antigen receptor for chemokines
(DARC), transferrin receptor 1 (TfR1) (also known as CD71), and CD98 [15,39–41]. Comple-
ment receptor 1 (CR1) binds to P. vivax erythrocyte binding protein RII [42] but has not yet been
shown to mediate P. vivax invasion. DARC binds to P. vivax Duffy binding protein (PvDBP)
[43,44], and many studies have contributed to the development of PvDBP as the leading
P. vivax vaccine candidate. Using DARC-negative cells, malaria parasites can still attach to
RBCs but do not form a tight junction, showing that DARC interaction is most likely involved in
tight junction formation. Genetically and phenotypically Duffy-negative individuals can still be
infected by P. vivax, indicating the possibility of other ligand–receptor interactions required
for P. vivax invasion and/or that the globin transcription factor 1 (GATA-1) SNP underlying

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Figure 2. Receptor–adhesin pairs in Plasmodium vivax targeting of nascent reticulocytes. This figure illustrates the
crucial interactions between P. vivax merozoite ligands (such as PvDBP, PvRBP2b, and PvRBP2a) and specific host receptors
– Duffy antigen receptor for chemokines (DARC), transferrin receptor 1 (TfR1), and CD98 – showing how these ligands target
and facilitate the identification and invasion of nascent reticulocytes (refer to the Glossary for definitions of these proteins).
The structural biology of these interactions has been reviewed and illustrated elsewhere [180]. There is potential for these
and other merozoite ligands to engage with other, unlisted reticulocyte receptors, underscoring the complexity of these
interactions. At present, the molecular mechanism(s) for P. vivax invasion have not been fully elucidated, and while these
host–pathogen interactions are shown as individual invasion pathways, future experimental evidence may show that they
work in the same invasion pathway during different steps of invasion. This figure was created using BioRender.

Fy-negativity does not entirely prevent Fy expression [45]. Indeed, recent studies show that, in
Duffy-negative individuals, erythroid precursors transiently express Duffy antigen, and can sup-
port P. vivax infection [45,46]. Invasion of these erythroid precursors, physiologically confined
to the bone marrow but present in the spleen in chronic vivax infection [19], may explain the
persistence of low-density P. vivax infections in Duffy-negative populations across sub-
Saharan Africa [47]. Amplification of PvDBP in the African P. vivax genome is a possible adap-
tation to enhance invasion in Duffy-negative populations [48,49] and may also enable P. vivax
to evade host antibodies in Duffy-positive populations [50]. In addition, CR1 binds to PvEBP-RII
irrespective of the presence of DARC on erythrocytes [42], and this may prove to be an alter-
native invasion pathway in Duffy-negative populations.

P. vivax reticulocyte binding protein 2b (PvRBP2b) binds toTfR1 (CD71) to mediate a critical
merozoite invasion pathway into reticulocytes [15,39]. Notably, TfR1 is abundant on young retic-
ulocytes, diminishing as they mature [17]. P. vivax invasion is significantly inhibited in the presence
of TfR1 mutant cells, showing that TfR1 is a critical host factor for entry into reticulocytes [15].

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Anti-PvRBP2b mouse monoclonal antibodies resulted in a reduction in parasite invasion using

Thai and Brazilian clinical isolates [15]. Naturally acquired human antibodies against PvRBP2b in-
hibit its interaction with TfR1 and binding to reticulocytes, and show different structural mecha-
nisms for inhibition, including either steric hindrance with TfR1-Tf or with the reticulocyte
membrane [51]. CD98 also binds to parasite ligand P. vivax reticulocyte binding protein 2a
(PvRBP2a) [40]. Anti-CD98 antibodies block P. vivax invasion in clinical isolates, though not to
as complete an extent as an anti-DARC antibody [40]. It is clear that P. vivax uses multiple inva-
sion pathways for entry into RBCs [52], similar to its counterpart P. falciparum [53], and that par-
asite ligands may interact with multiple reticulocyte receptors. At present, the full molecular
mechanism for P. vivax has not been elucidated and while we may refer to these host–pathogen
interactions as individual invasion pathways, it may be that future experimental evidence will show
that they work in the same invasion pathway during different steps of invasion. It will be important
to understand the contribution of each of these invasion pathways for the rational design of
P. vivax vaccines. While invasion by Goan P. vivax isolates was inhibited by either the cytokine
melanoma growth-stimulating activity (MGSA) or a monoclonal anti-TfR1 antibody to inhibit
PvDBP/DARC and PvRBP2b/TfR1 interactions, respectively, IC50 values ranged from four- to
12-fold for the single interventions. However, when the interventions were applied in combination,
there was a significant increase in invasion inhibition, suggesting that targeting both TfR1 and
DARC invasion pathways simultaneously would represent an attractive vaccine design [52].

Relapse from hepatic hypnozoites

Relapse, recurrent parasitemia from previously acquired latent parasites (hypnozoites) in the liver,
is a cardinal feature of P. vivax, although other human-only (Plasmodium ovale) and zoonotic
(Plasmodium cynomolgi, Plasmodium fieldi) species also relapse [54]. As outlined below, relaps-
ing infection is a major contributor to severe disease and death, particularly in high-transmission
areas [5–7].

The patterns and determinants of relapse have been reviewed elsewhere [4,55,56]. Traditionally,
relapses were thought to be geographically strain-specific, with fast-relapsing ‘tropical’ strains
contrasted with slow-relapsing ‘temperate’ strains. This is an over-simplification, with long-
latency phenotypes being more widespread than previously thought, and demonstrated by ob-
servations that injecting fewer sporozoites could transform fast- into slow-relapsing strains [55].
The Darwinian drivers for such adaptation include the need of the parasite to maintain itself in
small host populations, and where seasonal transmission is limited to only a few warmer months
when vector mosquitoes are present. These adaptations have contributed to P vivax extending
far beyond the geographic range of P. falciparum and present huge challenges to malaria elimi-
nation efforts. The frequency of relapse is proportional to hypnozoite burden [57,58], which varies
markedly within vivax-endemic populations, resulting in 30% of patients having almost 70% of all
vivax recurrences [56]. Relapse risk varies by not just geographic variations in latency but also
transmission intensity, season, host immunity, age, and individual differences in infection risk,
host genetics, and previous chemotherapy [56], such that information from one population is
often difficult to apply to other groups [4,56]. Within a given population, relapse risk is also
compounded by temporary increases in risk, such as from intercurrent infections [56,59].

Hypotheses for hypnozoite commitment determining how many sporozoites immediately give rise
to hepatic asexual parasite development resulting in bloodstream parasitemia, and how many be-
come latent hypnozoites that trigger future relapses, fall into three categories [54]: (i) sporozoites
are genomically pre-committed to immediate asexual reproduction or latency, (ii) commitment oc-
curs only during the complex transmission cycle from mosquito to human, or (iii) commitment is
modified by the hepatocyte microenvironment. These three basic categories are not mutually

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exclusive and are difficult to distinguish [54]. Major progress in understanding hypnozoite biology is
being made using humanized mouse models and in vitro hepatocyte cultures for both P. vivax and
P. cynomolgi [60–65]. This includes the discovery of transcriptional and translational repression
mechanisms to maintain dormancy and proteolytic activity to sustain viability [33]. Epigenetic
mechanisms also modulate hypnozoite persistence [66,67]. Because of the difficulty in growing
blood stages of P. vivax, cultures used to infect mosquitoes are initiated from blood from clinical
cases. These isolates are heterogeneous, leading to reproducibility issues.

Most vivax infections (up to 90%) are attributed to relapses, which are often oligo-symptomatic
[5]. However, not all 8-aminoquinoline-preventable recurrences may be true relapses arising
from hepatic hypnozoite activation. Primaquine, activated through an enzymatic pathway present
in liver, bone marrow, and likely spleen [68], will also contribute to killing of asexual stages in the
vast extravascular reservoirs in the spleen and bone marrow [12,13,20,69], thus preventing their
release back into the circulation to precipitate recurrence [70]. The proportion of this hidden
asexual-stage release in accounting for what are currently attributed to relapses is unknown.
That aside, relapsing human parasites are usually the last non-zoonotic malaria parasites remain-
ing, at least partly (if not only) because hypnozoites can survive for a long time. Three years of no
indigenous transmission is necessary for a country to qualify for elimination certification [5].

What signals activate latent liver parasites, reliably communicating improved transmission oppor-
tunities? Plasmodium infection triggers vivax relapse, with greater risk following vivax malaria than
falciparum malaria [71] and following symptomatic more than asymptomatic infections [72]. Both
acute infections reflect recent mosquito activity and receptivity to ongoing transmission [55]. Non-
malaria infectious diseases such as typhoid or relapsing fever also precipitate vivax relapses, sug-
gesting that fever and hemolysis may be important factors [59]. Parasite-free blood transfusions
also increase the risk of relapse, indicating a key role of cell-free heme/hemoglobin in triggering
relapse [58]. Hypnozoites are destroyed by redox-active drugs, specifically 8-aminoquinolines
such as primaquine and tafenoquine [73]. Non-metabolizers of 8-aminoquinolines continue to re-
lapse, indicating that redox stress including decreased erythrocyte survival and increased heme
recycling in the liver are related to hypnozoite activation [73,74]. A functional spleen may also
modulate relapses, as asplenic persons have a high frequency of recurrent infections, probably
relapses [75].

Hidden parasite biomass and disease severity

It has long been recognized that disease severity of falciparum malaria is directly related to total
parasite biomass, especially sequestered parasite biomass within the microvasculature. Until re-
cently, it had been widely assumed that, with minimal microvascular sequestration, essentially all
vivax parasites circulated and were detectable in the peripheral blood. With parasitemia usually
<0.2% in peripheral blood and almost always <2%, this raised questions as to how P. vivax
could cause severe disease. Indeed, vivax disease severity has been correlated with circulating
parasitemia in only some studies [9,76]. The recent recognition of a large extravascular vivax par-
asite biomass has changed this paradigm. The ratio of splenic to circulating total parasite bio-
mass of 81 in chronic infection [13] is likely lower in acute malaria [13,36]. Nevertheless, the
evidence for a major non-circulating parasite biomass extends across the spectrum of disease,
from asymptomatic parasitemia [12,13] to moderately severe and severe disease [9,20,77]. In
a Malaysian study, whereas parasitemia was twofold higher in severe malaria, plasma PvLDH
as a measure of total biomass was sevenfold higher in severe compared to non-severe vivax
malaria [9]. In this same study, total body parasite biomass was associated with measures of sys-
temic inflammation, suggesting that extravascular parasite accumulation in the spleen and bone
marrow can mediate systemic inflammatory responses [9,77].

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Rheopathobiology
Altered host RBC deformability
In falciparum and knowlesi malaria, the deformability of both infected and uninfected RBCs
(iRBCs and uRBCs) is reduced, hindering unobstructed RBC movement within the microcircula-
tion [78]. By contrast, deformability of RBCs infected with P. vivax ring and trophozoite-stages is
enhanced [79]. Prior to invasion by P. vivax, the nascent CD71+ reticulocytes are larger, stiffer,
and more adhesive than mature RBCs [16,80]. Parasite invasion leads to rapid expulsion of retic-
ular material and adhesive surface receptors from the RBC, usually within 3 h [14]. This decon-
struction leads to reduced shear elastic modulus, reduced intracellular viscosity, and increased
deformability of the iRBC, enabling its unimpeded movement within the circulation [79].

The mechanisms by which P. vivax enhances the deformability of its host reticulocytes to match
that of normal RBCs are not fully understood. The infected 'reticulocyte’, which lacks reticular
matter, remains enlarged during P. vivax development. Notably, in fixed smears, reticulocytes
containing P. vivax trophozoites and schizonts appear larger in diameter compared to uninfected
reticulocytes or RBCs infected with other Plasmodium spp. [79]. This implies additional methods
by which P. vivax augments overall infected reticulocyte deformability, possibly by altering the
material properties of the RBC membrane skeleton to increase elasticity. During the second
half of the erythrocytic cycle, iRBCs lose their biconcave shape and become more spherical.
The geometry of a cell significantly impacts its rheological traits [80]. More spherical RBCs,
regardless of cytoskeletal or membrane properties, exhibit reduced deformability compared to
similarly sized but less spherical RBCs. Late-stage schizonts of P. vivax become highly spherical,
impacting their ability to move through narrow microcapillary beds [81]. The fewer circulating
mature schizonts seen in vivax malaria may be due to biomechanical constraints arising from
the spherical geometry [82] and rosetting [80,83], thus increasing trapping in the spleen [12,13]
and microvasculature [84].

In contrast to the enhanced deformability of ring and trophozoite-stage stage P. vivax-iRBC

(Pv-iRBC), uRBCs in vivax malaria have reduced deformability [85,86], though not as marked
as seen in falciparum malaria [78,86]. This likely contributes to the splenic trapping and con-
gestion with uRBCs seen in vivax malaria and vivax-associated anemia [17]. Reduced
deformability of uRBCs in vivax malaria may be related to heme-induced oxidative damage
[87], anti-RBC antibodies [86], or as-yet-unidentified parasite products [88].

Endothelial cell cytoadhesion

A major mechanism of pathology in falciparum malaria is the cytoadherence of iRBC to microvas-
cular endothelium and impaired blood flow to vital organs [3]. By contrast, this appears to be gen-
erally minimal in vivax malaria. A paucity of microvascular accumulation of Pv-iRBC has been
noted in vivax autopsy studies [89], although the effect of pre-mortem antimalarial treatment is
not known. While mature Pv-iRBCs are capable of adhering to endothelial cells in vitro [90], the
frequency is significantly less than with P. falciparum [91]. Unlike P. falciparum, P. vivax lacks
the var gene family, implying alternative mechanisms for cytoadhesion, such as VIR proteins
[92–94]. The spleen plays a major role in expression of parasite proteins involved in
cytoadherence [94]. Cytoadherence is observed with specific receptors and is, at least in part,
mediated by VIR proteins [91], but determining VIR ligands and their receptors is complex due
to the extensive repertoire of vir genes in the P. vivax genome. In chronic clinical infections, it
has also been noted that cytoadhesion of nascent reticulocytes to splenic sinus endothelial
cells is greater in vivax than falciparum malaria, suggesting that P. vivax products modify host re-
ticulocyte and/or endothelial cell adhesiveness in the spleen [13]. This may be a favorable adap-
tation, with the increased fragility of schizont-stage Pv-iRBC after successfully traversing narrow

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splenic endothelial slits [81,95] potentially accelerating rupture and increasing the likelihood of in-
vasion of adjacent cytoadhering static nascent reticulocytes in the sinus lumina [13].

Rosetting
In all Plasmodium species, iRBCs form clusters with uRBCs [96]. Rosettes in P. vivax are sta-
ble under shear stress, with binding mediated by the bric4 moiety of glycophorin C, favoring
normocytes over reticulocytes [97,98]. The inability of P. vivax to form rosettes with nascent re-
ticulocytes stymies the hypothesis that rosettes facilitate merozoite invasion. Recent work sug-
gests that the primary purpose of rosetting is to shield iRBCs from immune detection and
phagocytosis [99]. P. vivax rosetting has been associated with anemia in clinical infections
[100], and thus may contribute to greater splenic RBC retention and congestion [17].
P. vivax forms giant rosettes, a phenomenon absent in P. falciparum [96,100]. The mecha-
nisms underlying these giant rosettes and their role in pathology warrant further exploration.
P. vivax gametocytes also form rosettes with uRBC, which increase mosquito infectivity,
suggesting a role in favoring transmission [101].

Loss of RBCs
Anemia in vivax malaria results predominantly from loss of circulating uRBCs and, to a lesser ex-
tent, iRBCs [102]. Loss of circulating Pv-iRBCs occurs from the obligatory destruction upon
P. vivax schizogony, the loss of structural integrity of the host reticulocyte following infection by
P. vivax [95], and splenic retention of Pv-iRBCs with and without phagocytosis [12,13]. Neverthe-
less, anemia in vivax malaria is predominantly due to loss of uRBCs [102]. Past modeling, when all
parasites were assumed to circulate, proposed a frequently-quoted loss of 34 uRBCs for each
iRBCs [102,103]. However, the recent finding of the large hidden biomass of Pv-iRBCs in the
spleen and other extravascular niches [12,13,20] has negated such assumptions, and reassess-
ment of this proportion by updated modeling is required.

Loss of uRBCs results from both intravascular [104] and extravascular RBC loss [17], with the lat-
ter predominating. Several mechanisms likely contribute to loss of uRBCs. Microfluidic studies in
vivax malaria have shown increased fragility of uRBCs [81]. Depletion of uRBCs defenses against
oxidative damage has been reported in vivax malaria [102]. Induction of autoantibodies to uRBCs
in vivax malaria has been associated with enhanced phagocytosis of uRBCs [105].
Antiphosphatidylserine autoantibodies enhancing uRBC phagocytosis are higher in patients
with vivax malaria than falciparum malaria, and associated with admission and nadir hemoglobin
in both uncomplicated malaria and volunteer infection studies [86,106]. Loss of complement reg-
ulatory proteins (CRPs) on uRBCs in vivax malaria enhances complement-mediated phagocyto-
sis and also contributes to severe anemia [107].

Excessive spleen-related loss of predominantly uRBCs is a major cause of anemia in vivax ma-
laria. It has been estimated that up to 23.8% of total RBCs in P. vivax infection are retained in
the spleen, with splenic retention associated with reduced hemoglobin [17]. Biomechanical reten-
tion of uRBCs within the spleen, with or without accelerated macrophage clearance [17], likely
arises from rosetting [100], reduced deformability of uRBCs [86], likely increased splenic filtration
stringency, as well as the abovementioned immune-related processes [86,107]. Enhanced
cytoadhesion of uRBCs to laminin a5 within the spleen has been proposed as another mecha-
nism contributing to uRBC retention [108]. Opsonization of uRBCs in the spleen by extracellular
vesicles in vivax infections may also increase splenic uRBC phagocytosis [109].

Compounding RBC loss is reduced bone-marrow production resulting from dyserythropoieis

[20,110] and hepcidin-mediated functional iron deficiency [111], though bone marrow

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 Trends in Parasitology

reticulocyte response to anemia appears adequate in chronic P. vivax infection [17]. Further loss
of RBC after commencement of treatment is exacerbated by delayed parasite clearance from
suboptimal schizonticidal therapy [112] and from dose-dependent primaquine-induced hemoly-
sis in glucose-6-phosphate dehydrogenase (G6PD) deficiency [83], which can cause massive
and sometimes fatal intravascular hemolysis [89,113,114].

Microvascular pathology
Endothelial activation, a consistent independent predictor of impaired organ perfusion, severe
disease, and death in falciparum malaria [115], is even greater in acute vivax malaria, and likely
to be a key mechanism underlying organ dysfunction in severe vivax malaria [3]. Endothelial acti-
vation occurs very early in primary infection, often prior to symptoms and microscopy-detectable
parasitemia [116]. The degree of endothelial activation and impaired microvascular reactivity in
severe vivax malaria is at least as high as that seen in severe falciparum malaria, with both asso-
ciated with impaired tissue perfusion and vivax disease severity [9]. Microvascular pathology in
vivax malaria is also exacerbated by endothelial dysfunction, occurring as a result of impaired bio-
availability of L-arginine and nitric oxide [104], and degradation of the endothelium-protective layer
of glycocalyx lining endothelial cells, potentially caused by marked elevation of the endothelial ac-
tivation mediator angiopoietin-2 [117]. Thrombotic microangiopathy may also contribute to im-
paired microvascular perfusion in severe vivax malaria. The protease ADAMST13 is reduced in
proportion to vivax disease severity [9]. ADAMST13 cleaves von Willebrand factor (vWF)
multimers, but deficiency leads to ultralarge and prothrombogenic vWF multimers which in turn
lead to platelet aggregation, adhesion, and microvascular thrombosis.

Inflammatory response
Vivax malaria generally presents with a lower peripheral parasitemia than falciparum malaria
[9,118,119], indicating a lower fever threshold relative to circulating parasitemia. Circulating
P. vivax GC-rich DNA, in hemozoin or immune complexes, is a key inducer of inflammasome
assembly and fever-inducing cytokines [120]. Other inflammatory responses, such as cytotoxic
CD8+ cells, are elicited to a greater degree in P. vivax than P. falciparum because of its pre-
ferred tropism for MHC-I-expressing reticulocytes that, unlike mature RBCs, can present anti-
gen directly to CD8 + T cells [121]. Other inflammatory responses have been linked to
microvesicles derived from Pv-iRBC [122]. Neutrophil activation and low-density neutrophils
are increased in vivax malaria [123–125], with neutrophil type I interferon signaling likely con-
tributing to hepatocyte injury and potentially systemic vascular pathology [123]. Endothelial in-
flammatory mediators such as angiopoietin-2 are higher in uncomplicated vivax malaria relative
to falciparum malaria despite lower peripheral parasitemia [9,126], supporting the long-held
notion of a greater inflammatory response to P. vivax [127]. However, the recognition that
the large hidden extravascular biomass in white-cell rich organs likely mediates systemic in-
flammation [9,77] clouds the interpretation of studies suggesting a greater inflammatory re-
sponse based on circulating parasitemia alone, particularly those based on per-circulating
parasite quantitation of inflammatory mediators. A greater post-treatment inflammatory re-
sponse in vivax malaria than falciparum malaria has been reviewed previously, including its
role in post-treatment acute lung injury [127]. This evidence is further supported by a recent
longitudinal comparative study showing a greater rise in the inflammatory marker ferritin follow-
ing treatment of vivax malaria versus falciparum malaria [111].

Thrombocytopenia
Thrombocytopenia is common in vivax malaria [9,118,119], with degree of thrombocytopenia
associated with disease severity [9] and risk of death in children and adults, particularly in
those with concurrent severe anemia [128]. However, disease severity and death are not

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linked to bleeding, and the role of platelet consumption in causing pathology in vivax
monoinfections is not clear. The major mechanism of thrombocytopenia in vivax malaria ap-
pears to be binding of platelets to Pv-iRBCs, where they mediate parasite killing [118],
followed by clearance of RBC–platelet complexes in the spleen [118,129], with parasitized
RBC–platelet binding greater with P. vivax than with P. falciparum [118]. Thrombocytopenia
is also likely exacerbated by platelet consumption due to impairment of ADAMST13 activity
and increased vWF binding [9]. Reduced platelet production of the glycocalyx protectant,
sphingosine-1-phosphate, may contribute to endothelial and renal tubular glycocalyx degra-
dation in vivax malaria [117].

Importance of recurrent infections in morbidity and mortality

P. falciparum and Plasmodium knowlesi are more capable than P. vivax of causing severe and
fatal malaria with an untreated primary infection [3,130]. Much of the morbidity and mortality as-
sociated with P. vivax in medium- or high-transmission endemic areas is due to the cumulative
effects of recurrent and chronic infections [5–8,131]. Most recurrences are primaquine-
preventable [69], being either relapses originating from hepatic hypnozoite activation [132], or re-
sulting from release of primaquine-susceptible asexual parasites from extravascular reservoirs
into the circulation [3,13,70] (Figure 1). The major morbidity from recurrent infections is progres-
sive anemia, where there is failure of hematological recovery prior to the next recurrence
[5–8,133,134]. The odds of anemia with any given symptomatic P. vivax infection are significantly
greater in regions with short relapse periodicity [112]. Recurrent infections also contribute to pro-
gressive malnutrition [7,135], which itself leads to an increased risk of severe disease and fatal
outcome from a recurrent vivax infection [7,133,136]. The importance of recurrent infections lead-
ing to a cumulative risk of mortality is illustrated by a recent study from Papua, Indonesia, where
vivax malaria was a significant risk factor for representation to hospital [6]. While the risk of death
in hospitalized patients, mostly children, in a first admission was higher in falciparum malaria, the
risk of death on the third admission was almost twofold higher in those with vivax malaria [6].

Organ dysfunction syndromes

Acute respiratory distress syndrome (ARDS) is well described in acute vivax malaria [3]. Fatal
outcomes can occur [89,130,137,138] in up to 33–49% of those hospitalized with ARDS in en-
demic settings [10,139]. Evidence for accumulation of P. vivax in the pulmonary microvasculature
is modest [76,84]. Histological features typical of ARDS found at autopsy provide much stronger
evidence for an inflammatory etiology [89,137]. In a systematic review, half of the cases of acute
lung injury and pulmonary oedema commenced after the start of antimalarial therapy [8], suggest-
ing that parasite killing exacerbates alveolar-capillary inflammatory response and fluid leakage
[84].

Acute kidney injury (AKI) in vivax malaria is uncommon and usually non-severe Kidney Disease
Improving Global Outcomes (KDIGO) stage 1 [119]. Severe AKI is reported, mostly in case series
from India [8], but the extent to which P. vivax is causative or incidental to AKI from other causes is
unclear. The most frequent histopathological finding in a systematic review of 45 cases of vivax-
associated severe AKI was acute tubular necrosis, followed by cortical necrosis and thrombotic
microangiopathy [8]. In severe falciparum and knowlesi malaria, severe AKI is strongly associated
with intravascular hemolysis causing heme-related toxicity to renal tubules and glomerular endo-
thelium, acute tubular necrosis, and endothelial activation [115,140,141]. In the absence of
primaquine therapy, the magnitude of intravascular hemolysis in non-severe vivax malaria [9] is
similar to that in non-severe falciparum malaria [142], but is not higher in severe vivax malaria
[9]. By contrast, severe AKI resulting from primaquine-induced hemolysis in G6PD-deficient
vivax patients clearly results from heme-mediated toxicity and acute tubular necrosis

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 Trends in Parasitology

[89,113,143]. Endothelial activation is markedly increased in severe vivax malaria and likely con-
tributes to AKI [9].

Splenic rupture
Splenic rupture in acute malaria occurs more often in adults, who have lesser capsule elasticity than
children, and is more commonly in vivax than falciparum malaria [144], perhaps because splenomeg-
aly, and therefore intrasplenic tension, develops more rapidly in P. vivax infection than in P. falciparum
infection [145]. Splenic rupture can result in hypotension, severe anemia, and death [10,89,144].
Pathological examination of such cases shows uRBC congestion and red pulp expansion,
intrasplenic hemorrhage [144], with or without follicular and B cell hyperplasia [146], and capsular
stretching and tearing [144]. Chronic vivax malaria increases the risk of trauma-associated rupture,
with splenomegaly due to red pulp congestion with uRBCs and with loss of white pulp [17], presum-
ably increasing intrasplenic tension, capsular stretching, and vulnerability to intrasplenic bleeding.

Hypotension
Hypotension likely results from an increase in response to inflammation of vasodilators such as
kynurenine [147,148], but not from an excess of vascular NO, as this is reduced in vivax malaria
[104]. In severe falciparum malaria, marked increases in intravascular hemolysis cause NO
scavenging and increased basal vascular tone resulting in a low frequency of hypotension [149].
The lesser intravascular hemolysis seen in severe vivax malaria has been proposed as a mecha-
nism underlying the greater proportion of hypotension seen in severe disease in vivax malaria [104].

Coma
Coma is rare in vivax malaria [150] and, with most series not excluding comorbidities [8], the caus-
ative role of vivax infection remains unclear. P. vivax infection has been associated with impaired
cognitive development in children [151] and prolonged cognitive dysfunction in the elderly [152],
with possible mechanisms reviewed recently [152].

Placental dysfunction
Vivax malaria in pregnancy is associated with placental dysfunction as evidenced by a greater in-
cidence of preterm birth and low birthweight in neonates [153,154]. In contrast to falciparum ma-
laria, most studies show no parasite sequestration in the placenta, but adverse pregnancy
outcomes are associated with monocyte infiltrates, inflammatory responses, syncytial knotting,
increased placental barrier thickness, dysregulation of angiopoietin-1 and -2, and reduction in vil-
lous size and vascularity, all likely contributing to placental insufficiency [154–156].

Factors modifying risk of infection and disease

Parasite factors
Malariotherapy data from the early 20th century showed that certain strains of P. vivax resulted in
higher mortality in debilitated neurosyphilis patients than in others [157]. The parasite factors as-
sociated with altered virulence remain poorly understood. Particular P. vivax proteins carried in
extracellular vesicles are able to increase adhesion of P. vivax-infected reticulocytes to human
spleen fibroblasts, thereby potentially increasing parasite biomass [18]. The spleen also modu-
lates expression of parasite factors [94]. Complicated disease has been associated with poly-
clonal infections [158]. Vivax strains with inherent short relapse periodicity increase the risk of
anemia and, in many settings, severe malaria [5,6,10,112]. Infection of the parasite itself may
speculatively alter P. vivax biology and pathogenesis. The Matryoshka RNA virus-1 infects
some but not all P. vivax parasites, but to date, no other human Plasmodium parasites [159].
This vivax-specific virus may plausibly alter both parasite and host responses at each stage of
the vivax life cycle [159].

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Host factors
Age
This is an important risk factor for anemia [160] and severe and fatal malaria, with the highest risk
group for all-cause death following vivax malaria in high-transmission regions being infants, both
for early and delayed death [6,7]. This probably reflects inadequate immunity in this age group, an
inability to administer primaquine before 6 months of age, and their greater propensity to develop
multiple relapses [5]. Conversely, the greater frequency of infection in early life results in the earlier
acquisition of clinical immunity and a lower frequency of clinical disease in older children and
adults [161]. At the other extreme of age, older adults with more comorbidities are prone to
greater disease severity from a primary infection. Female sex is associated with a greater risk of
anemia [112], and higher risk of death from vivax-associated ARDS [139].

Genetic factors
These factors also alter the risk of infection and disease. Duffy (Fy)-negativity markedly reduces
the risk of symptomatic vivax malaria and parasite density, but as outlined above, does not ablate
risk of very-low-density subclinical infections in African populations [47]. The Duffy Fya+b– pheno-
type also reduces risk of clinical vivax malaria [162]. Alpha-thalassemia [163] and HbE-beta-
thalassemia [164] both increase the risk of vivax malaria, the latter possibly because of
reticulocytosis, though exact mechanisms are unknown. Southeast Asian ovalocytosis protects
against vivax malaria [165]. The lesser CR1 expression on RBCs in Melanesian populations
may contribute to the greater risk of severe anemia from P. vivax across the island of New
Guinea [127,134]. G6PD deficiency reduces the risk of vivax malaria [166,167], increases the
risk of anemia at the time of presentation [112], and when severity precludes clinician use of
primaquine, results in a greater number of relapses [5]. Patients with genetic impairment of
CYP2D6 activity have greater risk of primaquine failure, and therefore an increased number of
relapses and greater morbidity [73].

Acquired risk factors

These factors for severe and fatal vivax malaria include many comorbidities prevalent in vivax-
endemic regions. Comorbidities are likely to be underestimated as contributors to severity and
mortality in vivax malaria [3,10,89,127]. Malnutrition increases the risk of severe disease and
fatal outcomes [6,7,133,136]. Moreover, the relationship between vivax malaria and malnutrition
is clearly bidirectional, with chronic and relapsing vivax malaria also causing malnutrition
[135,168,169].

Coinfections may also increase vivax severity. In regions of high coendemicity, mixed P. vivax and
P. falciparum infections are associated with higher risk of severe anemia [134,170] and mortality
[6,171,172] than either infection alone. HIV, a risk factor for severity in falciparum malaria [173],
has also been linked to fatal vivax malaria [133], though more studies are needed to clarify this re-
lationship. Dengue coinfections are common and associated with an increased risk of bleeding
and dyspnea compared with vivax monoinfection [174]. Concurrent Gram-negative bacteremia
is common in vivax infections in India, including Salmonella typhi and Salmonella paratyphi
[175], which may contribute to the high frequency of vivax disease severity reported in this coun-
try [8,176]. Severe disease in vivax malaria has also been linked to concurrent bacteremia with
Streptococcus pneumoniae [9].

Chronic diseases such as diabetes increase the severity of falciparum malaria [177]; however, the
relationship between diabetes and vivax malaria is not clear. In low-endemicity Thailand, chronic
disease was sixfold more common in patients requiring hospitalization with P. vivax than
P. falciparum infection [130]. Acute and chronic comorbidities were found in the majority of

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vivax-attributable deaths in a Brazilian autopsy series [89], and each increased the risk of death Outstanding questions
from vivax malaria in a large prospective hospital series in Brazil and India [10]. Splenectomy Life cycle
markedly increases the risk of recurrent vivax malaria [75], and has been associated with severe What is the magnitude and architectural
vivax malaria [178], highlighting the dual role of the spleen, with its filtration and immune functions distribution of P. vivax biomass in the
modulating the risk of severe disease, at the same time as it is an organ harboring a huge reservoir bone marrow and spleen in acute and
chronic infections, relative to circulating
of replicating parasites that also contribute to morbidity and chronic infection. blood?

Pregnancy To what extent does erythropoiesis

occur in nonphysiological sites such
An increased risk of severe and fatal vivax malaria has been identified in pregnant women, with
as the spleen in P. vivax infection, and
severe malaria estimated by meta-analysis to occur in 4.1% of vivax infections in pregnancy [8]. how does P. vivax trigger this?
In a large prospective series, half of P. vivax-associated deaths in women were pregnant [10].
Complications associated with death in vivax malaria include ARDS [10,130], severe anemia, Do P. vivax gametocytes accumulate
in the skin to facilitate transmission?
AKI, and shock [10], although the causes of the increased risk of these complications in preg-
nancy are not well understood. What proportion of schizonts found in
the perifollicular zone and sinuses of
Concluding remarks the spleen are committed to become
gametocytes?
Recent advancements have significantly enhanced our understanding of P. vivax biology and path-
ogenesis in human infections. Notable findings include the extensive extravascular reservoirs and Do reticulocyte-derived exosomes me-
the endosplenic life cycle of the parasite within the reticulocyte-rich spleen. Improved knowledge diate intercellular communication during
P. vivax infection?
of parasite invasion pathways is crucial for the rational development of vaccines targeting
P. vivax. There is now a deeper understanding of how relapsing and chronic infections contribute Relapse
to morbidity, mortality, and transmission, as well as of the underrecognized impact of comorbidities Which factors activate hepatic
in exacerbating severe disease and mortality. Crucial pathobiological processes have been uncov- hypnozoites to cause relapse, and
ered, shedding light on mechanisms, including RBC loss and endothelial activation, that underpin by which mechanism(s)?

disease severity. However, many questions remain unanswered (see Outstanding questions) and How do the genome and epigenetic
addressing these will significantly bolster efforts towards the prevention, management, and even- elements control relapse periodicity?
tual eradication of vivax malaria.
What proportion of (i) 8-aminoquinoline-
preventable recurrent acute infections
Acknowledgments and (ii) chronic infections arise from
We regret that not all relevant primary data articles, particularly from the informative older literature, could be cited in this re- true hypnozoite relapse versus release
view because of space constraints and recency criteria imposed by the journal. Supported by National Health and Medical of asexual parasites from vast extravas-
Research Council Investigator Grants to S.K. (#2025376) and W-H.T. (#2016908). cular reservoirs, thereby causing de-
layed recrudescence?

Declaration of interests
How does the spleen modulate vivax
G.D.S. is an unpaid consultant to GSK on tafenoquine. All other authors declare no competing interests. relapses?

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