Pathogenesis of Severe Malaria

Agung Nugroho
Division of Tropical & Infectious Disease
Internal Medicine Department
Sam Ratulangi University Manado
 Outline

• Introduction
• Epidemiology
• Pathogenesis of severe malaria
– Overview
– Sequestration of infected erythrocytes : role of PfEMP-1
– Role of cytokines
– Endothelial activation : Role of Angiopoeitin
– Endothelial dysfunction : Role of nitric oxide
• Summary
 Introduction

• Althrough recently the incidence of malaria has drop

significantly , still it takes ≈ 600.000 of lives annually
• Severe malaria is relatively a rare (1-2 % of malaria cases), but
responsible to almost all malaria – related death ( 90 % )
• Even with effective antimalaria and supportive treatment ,
mortality of severe malaria is still high : 10 – 20 %
• Knowledge of pathogenesis of malaria still evolving and not fully
understood
• Understanding the pathogenesis of malaria is important to
develop an effective adjunctive therapy of severe malaria

Wassman SC et al. Am J trop Med Hyg 2015 ; 93 ( Suppl 3 ) : 42

 Epidemiology

• In high- transmission area / stable endemic :

– Severe malaria commonly affect young chidren < 5
year old
– Older children and adult have already immune,no
severe malaria
– 90 % of the world,s severe and fatal malaria affect
young children in Sub-Sahara Africa
• In low – transmission area / unstable endemic :
– severe malaria afffect both children and adult

WHO. Severe malaria. TM & IH 2014 ; 19 ( Suppl. 1 ) : p. 7

 Epidemiology

• Risk factors of severe malaria :

– Children age < 5 year
– Elderly
– Immunocompromized : HIV, transplant
– Non-immune persons : migrant, travellers
– Pregnant women
 Pathogenesis of severe malaria : overview

• Complex ! , multifactorial
• Not fully understood, but progress
• Factors contribute to severe malaria :
– Host factors : immunity, genetics
– Parasite factors : toxin, drug resistance gene
antigenic variations, etc.
– Social and demografic factors
Pathogenesis of severe malaria : overview
 Pathogenesis of severe falciparum : overview
• Cytoadherence : sequestration of infected erythrocyte ( IE )
within the microvasculature of various organs leading to
microvascular obastruction
• Activation of immune cells and released of proinflammatory
cytokine
• Dysregulation of coagulation pathway
• Impairment of angiopoietin-2 – Tie2 system
• Impairment of nitric oxide ( NO ) bioavailability
• Rosetting and clumping microvascular obstruction
• Others : low heme-oxygenase-1 and glutamin
low Heat Shock Protein ( HSP ) - 70
impairment of angiotensin system.
 Pathogenesis of severe malaria : overview
Malaria infection

Cytoadherens inflammation Coagulation

activation

microvascular Endhothel activation

obstruction and dysfunction

tissue & organ dysfunction

Janet S, Alison GC. Frontiers in cellular and infection microbiology 2014 ; 4 : 4

Pathogenesis of severe malaria : overview
 pathogenesis of severe malaria : overview

Both sequesteration of IE and inflammation are important

 Cytoadherence

• Sequesteration of IE to endhothelial cell ( Cytoadherence ) is

pathological hallmark

Cytoadherence

Microvascular obstuction Endothelial dysfunction

Reduced blood flow

Pro-inflammatory cytokine
Vasoconstriction
Coagulation disorders
Tissue hypoxia &
Damage BBB vascular leakage oedema
ischaemia

Organ dysfunction
 Mechanisms of Cytoadherence

• Cytoadherence involve binding of parasite protein PfEMP1 to

many adhesion molecule on endothel cell : ICAM-1, CD36,
VECAM, ELAM, CSA
• PfEMP1 anchored into “ knob like particle “ on IE
• PfEMP1 encode by var genes ; there is 60 var genes in each
parasites, So PfEMP1 is highly polymorphic that consists of
many variant /strain
• Only 1 var gene is active in every parasite replication cycle
• PfEMP1 classified into 3 subfamily : group A, B, C, and many
strains like var1csa ; var2csa, others var ( IT4var09 )
 Mechanisms of cytoadherence

Severe malaria results from binding of PfEMP-1 with

endothelial adhesion molecules
 Mechanisms of Cytoadherence

• Recently severe malaria is linked to distinc PfEMP1

variant :
– Group A : CIDRα-1
– Group B / A : DC 8 , DC13
• PfEMP-1 group A and group DC- 8, DC 13 bind to
Endothelial Protein C- Receptor ( EPCR )
 Structure and function of PfEMP1

Characteristic of P. falsiparum
IE : protrusion particle on the
surface of erytrocyte called
knob that consists of Pf-
EMP1 and other proteins

Smith JD. Mol Biochem parasitol 2014 ; 195 (2) : 82-87

 Structure and function of PfEMP1

Severe malaria develop when there is PfEMP1 variant DC8 or DC 13 that bind to EPCR

Smith JD. Mol Biochem parasitol 2014 ; 195 (2) : 82-87

 Sequestration and severe malaria

PfEMP1- DC4 + ICAM1 only : sequesteriation (+), no severe malaria ?

PfEMP-1- DC8 or DC13 + EPCR : sequesteration (+) , severe malaria ( + )
PfEMP1 + ICAM1 + EPCR : heavy sequesteration , severe malaria ( + )
PfEMP1 group B / C + CD36 : sequesteration (+) , no severe malaria

Aird WC, Mosnear LO, Fairhurst RM. Blood 2014 ; 123 (2) : 163 - 167
 Pathogenesis of severe falciparum malaria :
Role of EPCR, protein C

• EPCR is natural ligand to protein C ( PC ) and activated protein

C ( APC )
• Function of Protein C : anticoagulant, antiinflammation
support expression of APC
• Function of APC : antiinflammation
antiapoptosis
protection of endothelial barrier function
• If EPCR bind to PfEMP1 instead of PC / APC, may cause :
inflammation and disminish of local blood flow

vascular dysfunction ; vascular leakage, edema

ischaemia

organ failure : coma, renal failure, lung edema, etc

 Pathogenesis of severe falciparum malaria :
Role of EPCR, protein C, APC

TM = thrombomodulin PC = protein C EPCR = endothelial protein C receptor

IIa = thrombin APC = activated protein C PAR-1 = protease-activated receptor-1
 Pathogenesis of severe falciparum malaria :
Role of EPCR, protein C, APC

Smith JD, Rowe JA et al. Cell Microbiol 2013 ; 15 ( 12 )

 Role of inflammation in
the pathogenesis of severe malaria

• Results from many studies had shown the evidence that

inflammation plays an important role in the pathogenesis of
severe malaria and death
• Inflammation caused by pro- inflammatory cytokines
triggered by malaria toxins : Glycosylpphosphotidylinositol
(GPI ) , parasite DNA in hemozoin
• Important : balance between pro – and anti-inflammation
cytokines
• There are conflicting results of which cytokines are crucial to
the development of severe malaria
 Cytokines related to severe malaria

Pro-inflammation Anti-inflammation
• TNF-α • IL-10
• IL-1RA • TGF-β
• IL-6
• IFN-ϒ
• IL – 12
• Chemokines ( IL-8, RANTES,
eotaxin )
• MIP, MCP
• HMBG1
 Role of Inflammation in severe malaria

• Inflammation may cause :

– Cellular dysfunction due to hypoxia :
• Mitochondria unable to use available oxygen cause
hyperlactatemia
• Mitochondria starved of oxygen due o inflammatory
cytokines indirectly limit the suply of oxygen to cells /
mitochondria
– Inflammatory cytokine cause blood elements ( leucocytes
and platelet , IE ) to adhere to endothelium through
increased expression of adhesion molecules like ICAM-1,
VCAM-1
– Inflammatory cytokines induced cellular apoptosis
Clark IA et al. Malaria journal 2006 ; 5 : 85
 Early production of IFN-ϒ and late production of IL-10 : resolution

Early production of IL-10 might reduced IL-12 cause low IFN and high TNF : severe malaria

Hassan DA et al. Khartoum Medical Journal 2010 ;3(1) : 373 - 376

 Endothelial activation and dysfunction

• Marker of endothel activation : von Willebrand factor

angiopoietin- 1 , Ang-2
• Marker of endothel dysfunction : vasodilation
nitric oxide ( NO )
• Function of angiopoietin-1 : vascular development
endothel cell survive
vascular assembly and maturation
• Function of angiopoietin-2 : antagonist to Ang-1
proinflammation : TNF, leucocyte
• Ang-1 & Ang-2 bind to Tie-2 receptor that expresion selectively
by endothelial cell and monocytes
Angiopoietin – Tie 2 system
 Pathophysiology of severe falciparum malaria :
role of angiopoietin – Tie 2 system

Incresed Angiopoietin-2 that bind to tyrosine kinase Tie- 2 receptor might

enhanced inflammation triggered by TNF
 Pathophysiology of endothelial dysfunction

Kim H, Higgins S et al. Current opinion in hematology 2011 ; 18 : 177 -180

 Endothelial dysfunction :
role of nitric oxide
• NO functions :
– Inhibiting Weibel – Palade body exocytosis ( Ang-2 released )
– Increasing Ang-1 expression
– Decrease endothelial expression of ICAM-1, VCAM-1, thus
inhibit cytoadherens
– Inhibit TNF production dampens inflammatory respons
– Inhibit procoagulant activity of endothelial cell
– Inhibit platelet aggregration dampens thrombosis
– Antimicrobial including killing P. falciparum
– Regulation of vascular tone and blood pressure
 Role of nitric oxide

• In severe malaria : low level of NO

• Low NO in severe malaria caused by low level of L- arginine
due to excess arginase enzym produced mainly by
plasmodium
• Impact of Low NO : vasoconstriction
inflammation
NO
Endogen iNOS
- Body protein degradation arginin parasite
- Glutamate citrulline arginase-1 IL-4 , 10,13
Exogen : diet hemolysis RBC

Ornithine + urea
Role of nitric oxide
 Role of Roset & clumping

• Roset : binding IE with uninfected erythrocyte

• Clumping : binding IE with platellets
• Both process have been associated with SM and CM
but maybe its role is less prominent than
cytoadherence
• Mechanisms of roset :
– Binding Pf-EMP1 of IE with CD36 of uninfected erythrocyte
, also involve IgM, α2-macroglobulin
– Binding of RIFINs of IE with antigen blood group A of
erythrocytes
RIFINs : repetitive interspresed family of proteins
 Roset & clumping

• Mechanisms of clumping :
– Binding Pf-EMP-1 of IE with CD36 , P-selectin and globular
C1q receptor (gC1qR )
• Roset and clump may cytoadhere to microvascular
endothelium
• Roset and clumping might enhance cytoadherence and
blocking microvascular
 Pathogenesis of severe vivax malaria

• Severe vivax malaria is less compare to severe falciparum

malaria
• P. vivax infected only reticulocytes no hyperparasitemia
• Most cases : severe anaemia , ARDS, coma ( rare )
• P. vivax have little if any sequesteration, roset,
autoaggregation / clumping
• P. vivax infections have lower pyrogenic threshold ( 180 vivax
parasites/uL compare to 1000 falciparum parasites / uL )
• Severe vivax malaria have higher inflammation due to higher
pro-inflammatory cytokines : IL-6 and IL-`10 were higher than
P. falciparum
 Pathogenesis of severe vivax malaria

• Cause of severe inflammation :

– Unknown
– Pv-GPI is stronger cytokines inducer than Pf-GPI ?
– P. vivax cytoadherence and roseting mediated by gen VIR ?
– A lipid fraction of cholesterol and triglyceride is strong
cytokines- inducer
 Summary

• Pathogenesis of severe malaria is complex and not fully

understood
• There are 2 important components :
– Mechanical theory : mechanical microvascular obstruction
due to IE sequesteration
– Cytokines theory : imbalance of pro and anti-inflammatory
cytokines leading to cellular / tissue injury
• Both cause endothelial activation ( high Angiopoeitin – 2 ) and
dysfunction ( low NO ) leading to organ dysfunction
• In severe falciparum malaria : Sequesteration is a hallmark
pathological events
• In severe vivax malaria : inflammation is important
Thank you