Colloquium Digital Library of Life Sciences

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1 Malaria

2 ii Colloquium Digital Library of Life Sciences This e-book is an original work commissioned for the Colloquium Digital Library of Life Sciences, a curated collection of time-saving pedagogical resources for researchers and students who want to quickly get up to speed in a new area of life science/biomedical research. Each e-book available in Colloquium is an in-depth overview of a fast-moving or fundamental area of research, authored by a prominent contributor to the field. We call these resources Lectures because authors are asked to provide an authoritative, state-of-the-art overview of their area of expertise, in a manner that is accessible to a broad, diverse audience of scientists (similar to a plenary or keynote lecture at a symposium/meeting/colloquium). Readers are invited to keep current with advances in various disciplines, gain insight into fields other than their own, and refresh their understanding of core concepts in cell & molecular biology. For the full list of available Lectures, please visit: All lectures available as PDF. Access to the Collection is free for readers at institutions that license Colloquium. Please info@morganclaypool.com for more information.

3 iii Colloquium Series on Integrated Systems Physiology: From Molecule to Function to Disease Editors D. Neil Granger Louisiana State University Health Sciences Center Joey P. Granger University of Mississippi Medical Center Physiology is a scientific discipline devoted to understanding the functions of the body. It addresses function at multiple levels, including molecular, cellular, organ, and system. An appreciation of the processes that occur at each level is necessary to understand function in health and the dysfunction associated with disease. Homeostasis and integration are fundamental principles of physiology that account for the relative constancy of organ processes and bodily function even in the face o f substantial environmental changes. This constancy results from integrative, cooperative interactions of chemical and electrical signaling processes within and between cells, organs and systems. This ebook series on the broad field of physiology covers the major organ systems from an integrative perspective that addresses the molecular and cellular processes that contribute to homeostasis. Material on pathophysiology is also included throughout the ebooks. The state-of the-art treatises were produced by leading experts in the field of physiology. Each ebook includes stand-alone information and is intended to be of value to students, scientists, and clinicians in the biomedical sciences. Since physiological concepts are an ever-changing work-in-progress, each contributor will have the opportunity to make periodic updates of the covered material. Published titles (for future titles please see the website,

4 iv Copyright 2013 by Morgan & Claypool All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopy, recording, or any other except for brief quotations in printed reviews, without the prior permission of the publisher. Malaria Lead Author: Juliana Carvalho Tavares ISBN: paperback ISBN: ebook DOI: /C00091ED1V01Y201309ISP045 A Publication in the COLLOQUIUM SERIES ON INTEGRATED SYSTEMS PHYSIOLOGY: FROM MOLECULES TO FUNCTION TO DISEASE Lecture #45 Series Editor: D. Neil Granger, LSU Health Sciences Center, and Joey P. Granger, University of Mississippi Medical Center Series ISSN ISSN X print ISSN electronic

5 Malaria Juliana Carvalho Tavares Universidade Federal de Minas Gerais COLLOQUIUM SERIES ON INTEGRATED SYSTEMS PHYSIOLOGY: FROM MOLECULE TO FUNCTION TO DISEASE #45 &C M MORGAN & CLAYPOOL LIFE SCIENCES

6 vi ABSTRACT This ebook describes the pathogenesis of malaria and the major consequences of the parasitism to the vertebrate host. Malaria is one of the oldest infectious diseases of mankind, which still exerts a high burden on human health and society. It is caused by parasites of the genus Plasmodium, and transmitted by Anopheline mosquitoes. Despite several decades of intensive control efforts, malaria remains widely distributed with an estimated 3.3 billion of the world s population at risk of infection. The malaria life cycle is extremely complex and the blood stage parasites are responsible for all the symptoms and pathology of malaria. Because of this strict association between the parasites and red cells, there are numerous consequences to the host s blood extending far beyond the direct effect of parasitized RBCs. Chapter 1 of the ebook summarizes the different clinical and pathogenic features of the disease, comparing the disease caused by Plasmodium falciparum and Plasmodium vivax, the two prevalent malaria species. This chapter is the basis of the remaining chapters, in which the different mechanisms of the pathogenesis of malaria are discussed in greater depth. In this direction, in Chapter 2 we discuss the role of host and parasite genetic diversity, which determines the malaria pathogenesis. Chapter 3 discusses the immune mechanisms associated with malarial anemia, a multifactorial clinical outcome which has not been fully addressed. In Chapter 4 we focus on the immune response associated with parasite control and microvascular damage. Chapter 5 presents information about epidemiology, clinical presentation, diagnosis, and management of cerebral malaria (CM), the most severe neurological complication of Plasmodium falciparum infection. Understanding how an intraerythrocytic parasite, which remains within the vascular space of the brain, causes profound neurological effects will be explained in Chapter 6, which presents the interesting mechanism of interaction between infected red blood cells (RBC) and the blood brain barrier (BBB). Finally in Chapter 7 we conclude with a discussion of the mechanisms of persistent cognitive and memory impairment associated with cerebral malaria. We hope that this ebook will be useful for students, professionals and researchers by providing an up-to-date summary of the factors that determine Plasmodium-associated morbidity and severe disease. KEYWORDS malaria, cerebral malaria, pathogenesis, immune system, blood brain barrier, neurologic impairment

7 vii Contents 1 Malaria Pathogenesis: Fernando Fonseca de Almeida e Val, Wuelton Marcelo Monteiro, and Marcus Vinícius Guimarães de Lacerda Parasite Biomass Inflammatory Response Infected Erythrocytes: Deformability/Rosetting Endothelial Alterations Cytoadherence/Sequestration Clinical Complications Anemia Thrombocytopenia/Coagulation Disorders Jaundice Metabolic Complications Pregnancy-Related Malaria Respiratory Distress and Lung Injury Neurological Syndrome and Coma Acute Renal Failure Algid Malaria/Shock Atypical Manifestations Comorbidities Possible Mechanisms to Explain Plasmodium Immunopathogenesis: The Role of Parasite and Human Host Genetic Diversity: Ricardo Luiz Dantas Machado Plasmodium falciparum Related Pathogenesis Plasmodium vivax related pathogenesis Cytoadherence and Virulence of Plasmodium Knowlesi Malaria Concluding Remarks

8 viii 3 Malarial Anemia: A Multifactorial Hematological Outcome: Luiza Carvalho Mourão, Thiago Castro Gomes, and Erika M. Braga Introduction Anemia and Parasite Biomass are not Associated Rosette Formation and Cytoadherence The Participation of Inflammatory Cytokines The Involvement of Nitric Oxide (NO) in Anemia Phagocytosis of Malaria-Infected and Non-Infected RBCs Complement Proteins and Anemia The Possible Role of Autoantibodies Concluding Remarks Immune Response to Malaria Blood Stage: Onesia Cristina Oliveira Lima and Juliana Carvalho Tavares Introduction Mouse models for malaria research Life Cycle of the Malaria Parasite Dendritic Cells CD4 T Cells NK (Natural Killer) Cells CD4+CD25+Foxp3+ regulatory T Cells γδ T-cel Monocite/macrophages B Cells Concluding Remarks Human Cerebral Malaria and Experimental Models: Leonardo J. M. Carvalho and Yuri C. Martins Introduction Cerebral Malaria: Epidemiology, Clinical Presentation, Diagnosis and Management Pathogenesis Adjunctive Therapies Animal Models of CM Conclusion

9 ix 6 Blood-Brain Barrier Disruption in Cerebral Malaria: Marcelo Limborço Filho and Juliana Carvalho Tavares Introduction Healthy Blood-Brain Barrier: Structure and Function How is it Possible to Study BBB Permeability? Rupture of the BBB in CM What Mechanisms Might Underlie BBB Dysfunction in Cerebral Malaria? PRBC Sequestration and Interaction with BBB Consequences of PRBC Adhesion on Endothelial Cell (BBB) Apoptosis of Endothelial Cell Blood Brain Barrier Inflammatory Mediators Leukocyte and Platelet Recruitment How Can Platelets Modulate EC-PRBC Interaction and Subsequent Sequelae? Consequences of BBB Rupture on Astrocytes and Microglia Concluding Remarks Persistent Cognitive and Memory Impairment after Cerebral Malaria: Molecular Mechanisms and Pre-clinical Models of Adjuvant Therapy: Patricia Alves Reis, Tatiana Maron-Gutierrez, Hugo Caire de Castro Faria Neto, and Guy Zimmerman Introduction Cognitive Impairment in Cerebral Malaria What Do We Know So Far? Neuroinflammation and Cognitive Impairment in Cerebral Malaria Mechanisms of Memory Impairment after Cerebral Malaria Therapeutic Approaches to Tissue Damage Associated to Cerebral Malaria Concluding Remarks Bibliography

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11 1 CHAPTER 1 Malaria Pathogenesis Contributing Authors Fernando Fonseca de Almeida e Val, Wuelton Marcelo Monteiro, and Marcus Vinícius Guimarães de Lacerda Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Amazonas, Brazil 1.1 PARASITE BIOMASS In malaria caused by Plasmodium falciparum (Pf ), the parasite has no specific predilection for any stage of circulating red blood cells (RBCs) and therefore invades all RBC stages irrespective of their age. This is one of the main reasons why in this type of malaria, parasitemias are usually high. If no treatment is initiated or if the host immune response is not effective, parasite biomass increases, eventually leading to severe disease and death. Elevated parasite biomass is a major independent mortality risk factor in this type of malaria, as first stated by Bignani and Marchiafava, the two Italian pathologists who described autopsied cases of severe Pf malaria [1]. However, malaria caused by Plasmodium vivax (Pv) usually presents with very low parasitemias, generally <100,000 parasites/ mm 3 of blood, due to the predilection for young RBCs (majorly reticulocytes). Usually parasitemias in vivax malaria do not exceed 2% of circulating RBCs. Although reports of high parasitemia in severe vivax malaria presentation have already been reported, ranging from 28,000 to 140,000/mm 3 [2 6], high parasite biomass as a severe disease marker for this species infection still needs further investigation [7]. A similar observation is valid for the presence of schizonts in peripheral blood, which is usually associated with high sequestered biomass and severity in falciparum malaria [8], but still an unexplored aspect in vivax. Another aspect of vivax malaria is that all stages of P. vivax infected red blood cells (Pv-iRBCs), principally the sexual forms (gametocytes), become available in blood circulation early in the course of infection, before clinical presentation and antimalarial therapy initiation. This differs from P. falciparum, in which gametocytes become detectable on peripheral circulation only in late phases of infection, due to adhesion of earlier stages in the bone marrow and therefore, after beginning of symptoms, which has an important impact on transmission intensity and control/eradication strategies.

12 2 MALARIA 1.2 INFLAMMATORY RESPONSE The intensity of host response to malaria disease varies with the species causing the infection. Vivax malaria has greater inflammatory response per parasitized erythrocyte than falciparum malaria, which is evidenced by vivax lower pyrogenic threshold (level of parasitemia associated with fever). Some reasons are hypothesized and involve possible differences in malaria parasite-derived toxins, which includes glycosylphosphatidylinositol (GPI) and parasite DNA associated with hemozoin. These malarial toxins differ in structural composition between P. vivax and P. falciparum and it is suggested that they may participate in the pyrogenic response through toll-like receptor-9 (TLR-9) stimulus, which was first attributed to hemozoin itself [9, 10]. By analyzing the relationship between host response and parasitemia of 125 patients with malaria (97 P. falciparum, 22 P. vivax and 6 P. ovale), Hemmer and colleagues found that host inflammatory parameters/parasitemia ratios were significantly higher in severe and mild P. vivax compared to P. falciparum malaria [11]. Yeo and colleagues observed similar alterations regarding endothelium-specific activation and inflammatory responses in uncomplicated infections regarding vivax and falciparum parasites [12]. It is suggested that host response helps to control parasite proliferation, and that failure to do so could lead to the increased parasitemias observed in falciparum malaria, with greater endothelial damage and specific organ damage which is not commonly seen in vivax malaria [11]. Host immune response also varies with clinical presentation in vivax malaria. Brazilian patients with severe vivax manifestations presented an imbalance of the pro-inflammatory markers TNF-alpha, IFN-gama and anti-inflammatory cytokine IL-10. In those patients, severe manifestations such as respiratory failure, severe anemia, renal failure, seizure and jaundice were present and not all survived. According to P. vivax infection severity, TNF-alpha, IFN-gama and IFN-gama/ IL-10 ratio increased with severity and showed a down-regulation behavior upon clinical recovery on those who survived [13]. Wider analysis of host immune response also showed marked imbalance of pro- and anti-inflammatory cytokines and oxidative stress (superoxide dismutase and heme oxygenase, SOD-1 and OH-1 respectively) among increasing severity groups of vivax malaria patients, and also the increasing complexity of interactions between markers according to the clinical groups [14]. The immune responses and exact mechanisms of tissue damage by inflammatory molecules and oxidative stress are not yet entirely understood. As reviewed elsewhere [15], it is suggested that the major mechanisms involved in malaria immunopathogenesis are disruption of nitric oxide (NO) metabolism with reduction of its bioavailability and impaired vasodilatation (which has been associated with pulmonary vasoconstriction in children with severe malaria anemia); release of inflammatory cytokines and adhesion molecules (ICAM-1) leading to rearrangements of cell cytoskeleton, which may contribute to vascular leakage (one of the main mechanisms that allied to

13 MALARIA PATHOGENESIS 3 inflammation could lead to cerebral edema and coma); and thrombosis and coagulation disorders which can generate clots that contribute to disruption of blood flow in microvasculature leading to metabolic acidosis and cerebral malaria. It is suggested that impairment of D M function, which represents alveolar-capillary gas transfer, after treatment in vivax and falciparum malaria, could be due to inflammatory response to parasite killing and reperfusion of pulmonary microvasculature [16]. 1.3 INFECTED ERYTHROCYTES: DEFORMABILITY/ ROSETTING One of the aspects involved in malaria pathogenesis, especially in severe forms, is whether the infected RBCs are able to adapt to reduced and narrowed environments from capillary beds in vital organs. In this particular aspect, both P. falciparum and P. vivax differ and consequences to clinical presentation and pathogenesis arise. P. falciparum infected erythrocytes have less ability to deform, and are thought to be more rigid than P. vivax-irbcs [17], a characteristic that can contribute to derangements of blood microcirculation in the former [18]. Mechanical microcirculatory impairment by irbcs has been confirmed by autopsy studies, but also observed in vivo with variable obstruction [19]. Non-infected RBCs also become less deformable [20] and the increased rigidity of irbcs and non-infected RBCs may be explained by the release of hemozoin during malaria infection, which could be responsible for erythrocyte membrane oxidation and consequent stiffness [21, 22]. Morphological, physiological and functional changes, with consequent reduction of flexibility in P. falciparum parasitized RBCs, also may be caused by the exportation of some specific proteins to the cell membrane surface [23, 24]. The age of the parasitized cells also seems to have an important relation to structural alterations, where RBCs infected with early stage parasites (rings) display minor changes in deformability proprieties, while late stage parasitized RBCs (trophozoites and schizonts) display more prominent modifications, leading to less ability to circulate in microvascular beds [24]. Microcirculatory obstruction leads to reduced blood flow, end-organ hypoxia, and function loss, which is the main pathogenic mechanism of severe falciparum malaria [25]. Contrary to Pf-iRBCs, deformability of Pv-iRBCs and non-pv-irbcs is increased [17, 26] and it is thought that this feature is an unlikely, or poorly contributing mechanism by which P. vivax causes severe disease. Another phenomenon that is linked to malaria pathogenesis and erythrocytes is the formation of rosettes. This feature is the ability of adhesion between uninfected and infected RBCs, and may contribute to vascular occlusion [27]. Together with decreased deformability of infected and non-infected RBCs mentioned before, it is thought to contribute to microvascular obstruction. Rosetting occurs with P. falciparum [28], contributes to anemia [29], and is associated with all clinical forms of severe falciparum malaria [30 32]. P. vivax-infected erythrocytes are also able to form rosettes [33 35]. A study conducted with Brazilian patients infected with P. vivax

14 4 MALARIA showed association between rosetting and anemia [36]. The exact mechanism by which rosetting may be related to severe vivax malaria is poorly understood. 1.4 ENDOTHELIAL ALTERATIONS Vascular endothelium dysfunction and activation play key roles in malaria pathogenesis, once one of the main pathogenic mechanisms includes impaired tissue perfusion by adherent parasitized erythrocytes with microvascular obstruction. In falciparum malaria, adhesive Pf-iRBCs disrupts endothelial homeostasis through increased adhesive promotion, coagulation disorders, inflammation, loss of endothelial barrier integrity (EBI) and impaired endothelium muscle tone control. Although less commonly described, endothelium injury in vivax malaria patient has been reported [37, 38]. Plasma concentrations of endothelial dysfunction markers, as Ang-2, ICAM and E-selectin, have been shown to be related to severe falciparum malaria [39], and as high as, or higher, in uncomplicated vivax compared to falciparum malaria in adults with similar parasite count [40]. Alterations in markers involved in coagulation disorders and thrombosis associated to falciparum malaria [41], have been reported in vivax malaria: ADAMTS13 deficiency and von Willebrand factor vwf elevation [42], thrombomodulin [43] and other pro-coagulant markers [44]. Loss of EBI is an important mechanism by which severe manifestations may occur: Ang-2 stimulation by increased expression of TNF may lead to expression of ICAM and might contribute to vascular leak and edema; reduced levels of ADAMTS13 lead to formation of large vwf multimers, which bind irbcs and platelets, leading to thrombi formation and microvascular obstruction; thrombim may lead to cellular cytoskeleton retraction and loss of EBI and also express inflammatory cytokines and adhesion molecules. These mechanisms are reviewed elsewhere [45]. Other mechanisms that seem to have an important role on severe falciparum pathogenesis are nitric oxide (NO) bioavailability reduction and concomitant impaired vasodilatation capacity [46 48]. Contributions to homeostasis loss through altered NO metabolism, intravascular coagulation, endothelium inflammation and platelet aggregation are not well understood in severe vivax pathogenesis. 1.5 CYTOADHERENCE/SEQUESTRATION The ability of infected RBCs to adhere to the vascular endothelium of vital organs is the hallmark of severe falciparum malaria pathogenesis. Through the expression of highly variable proteins on the surface of the infected erythrocyte, named P. falciparum erythrocyte membrane protein-1 (PfEMP1s), the irbc is able to bind to specific ligands expressed in the endothelium surface of venules and capillaries (CD36, intercellular adhesion molecule 1 ICAM1, platelet/endothelial cell adhesion molecule PECAM, heparin sulfate HS and chondroitin sulfate A - CSA) and become attached to the site. This allows the irbcs to sequester, avoid spleen passage, and trigger a series of homeostatic disturbances that may lead to severe disease. Pf-iRBCs sequester in various organs

15 MALARIA PATHOGENESIS 5 such as lung, brain, placenta, kidney, liver and microvasculature. This depends on host site receptor expression and var genes encoding PfEMP1s [49]. The inflammatory response seems to participate in the cytoadherence phenomena. Through the rupture of infected late stage erythrocytes, called schizonts, several components are released into the circulation, provoking an inflammatory response which in turn stimulates the expression of endothelial receptors involved in cell adhesion, and thus promotes higher cytoadhesion [50]. In malaria caused by P. vivax, if citoadhesion and sequestration is present, it is thought to happen in a degree that could not be responsible for severe forms as is reported from falciparum severe cases [51]. Evidence for P. vivax cytoadhesion is scarce. Carvalho and colleagues showed that Pv-iRBCs were able to cytoadhere in vitro to human lung endothelial cells HLECs, saimiri brain endothelial cells SBECs and to placental cryosections. [52]. In this study, Pv-iRBCs binded to the endothelial receptors ICAM1 and CSA with similar strength as Pf-iRBCs, but in a frequency 10-fold lower. Sequestration of parasitized RBCs in the spleen [53] and in the bone marrow may be higher in P. vivax, defying the old concept that the parasite s biomass is much lower than in P. falciparum. It is possible that more Pv parasites are sequestered in these lymphoid organs, source of distinct populations of reticulocytes. Major modifications triggered by this parasite in the spleen may lead to immunological alterations such as plasmoblasts hyperplasia [54]. 1.6 CLINICAL COMPLICATIONS P. vivax can cause severe or even fatal episodes, with incidence and mortality rates similar to those for P. falciparum and a broad clinical spectrum (Figure 1.1).

16 6 MALARIA FIGURE 1.1: P. vivax can cause a wide spectrum of clinical symptoms, ranging from single-organ affectation to multiple life-threatening organ failure. Used with permission from: Nature Medicine 17(1); 48-9, ANEMIA The World Health Organization (WHO) defines severe Pf malarial anemia as hemoglobin below 5g/dL in children and below 7g/dL in adults. Malaria-associated anemia is a greater burden in terms of frequency and severity in young children [55 58] and in pregnant women [59, 60]. The complication is also being more frequently associated with P. vivax. In the Brazilian Amazon, where P. vivax predominates, non-severe anemia frequencies can be as high as 80% in children and adolescents [61, 62].

17 MALARIA PATHOGENESIS 7 Anemia etiology is complex and is multifactorial in endemic areas (Figure 1.2). Major influencing factors are hemolysis, concomitant infection by intestinal helminthes, hemoglobinopathies, nutritional deficits, and many other factors summarized in Figure 1. Deficiency of glucose-6-phosphate dehydrogenase (G6PD) may predispose to severe hemolysis when the patient receives the hypnozoite-targeted antimalarial primaquine. That may also contribute to anemia amongst the affected population, with varying frequencies depending on ethnical background. As chloroquine resistance spreads, chronic infections, recrudescence, and delayed parasite clearance become important and may also contribute to enhance this complication [63]. Although several mechanisms seem to participate in the triggering of anemia in individuals infected with malaria, the mechanisms can be grouped into two main categories: intravascular (irbcs burst) and extravascular hemolysis (destruction and removal of cells from circulation) and diserythropoiesis. Malariotherapy retrospective data from the past showed that for every infected RBC in vivax malaria, nearly 30 non-infected RBCs are removed from circulation whereas in Pf, this number is approximately eight, showing that anemia is not solely the product of infected RBCs destruction [59, 64, 65]. The spleen plays an important role in developing anemia by clearance of infected and uninfected RBCs, through detection of mild erythrocyte membrane mechanical damage. There is scarce information on the effect of Pv upon erythropoiesis, despite the presence of the parasite in this milieu [66]. Two cases of Pv malaria revealed parasitized bone marrow smears in P. vivax blood smear negative patients, suggesting that erythroblast destruction by parasites may be an underlying mechanism of Pv related anemia [67]. Impairment of erythropoiesis in falciparum malaria may be possible due to inadequate erythropoietin formation [68, 69] and hypoxia of the bone marrow with suggested mechanisms of sinusoid obstruction by irbcs [70]. These mechanisms still lack in vivo evidence in vivax malaria despite studies showing impairment of erythropoiesis due to cytokine production, bone marrow phagocytosis and toxicity of the parasite in vivax malaria [70, 71].

18 8 MALARIA FIGURE 1.2: The ABO blood group system and Plasmodium falciparum malaria. Used with permission from: Cserti CM, Dzik WH. 2007, Blood.1;110(7): THROMBOCYTOPENIA/COAGULATION DISORDERS Although a frequent complication in malaria, thrombocytopenia by itself has never been regarded as a cause of death per se. It seems to be frequent in either Pf or Pv malaria, and some studies point to the negative correlation with peripheral parasitemia, suggesting that the reduced lifespan of platelets may be associated to antigen removal from the circulation. Thrombocytopenia occurs in 24 to 94% of malarial patients [72] and has had some unclear relation to malaria severity both in vivax and falciparum disease [73]. Thrombocytopenia is defined as platelet counts <150,000/μL, and severe thrombocytopenia as <50,000/μL. Pathogenesis of malaria thrombocytopenia is complex and as reviewed elsewhere [72], shows several features that are not entirely understood. Activation of the coagulation cascade could be partially responsible for the decrease in platelet count as well as splenomegaly. However, megakariocytosis and mega platelets release have been suggested as a compensation mechanism for low platelet counts due to peripheral destruction; platelet-targeted antibodies, and its removal from circulation, due to the binding of parasite antigens to its surface

19 MALARIA PATHOGENESIS 9 is another suggested pathway of thrombocytopenia, but lacks evidence on how autoimmune mechanisms could effectually cause this complication. Free radicals produced in increased oxidative stress during malarial infection is another feature and studies show negative correlations between platelet counts and platelet lipid peroxidase and positive correlation with anti-oxidant gluthatione peroxidase and superoxide dismutase activity (74). Platelet aggregation, endothelial activation and perturbation with ADAMTS13 deficiency and increased concentrations of active and ultra-large vwf, leading to coagulopathy, are other possible mechanisms by which thrombocytopenia may be induced [42, 72]. Recently, platelets from malarial patients were shown to be associated with increased phagocytosis by THP-1 cells [75] JAUNDICE As stated in the WHO Severe Malaria Treatment Guidelines (2010), clinical jaundice only becomes a feature of severe malaria if there is evidence of other vital organ dysfunction associated. Total bilirubin >3.0 mg/dl defines hyperbilirubinemia. Mostly adults are seen with jaundice and it is mostly seen in vivax as compared to falciparum disease [56, 73, 76]. Jaundice as part of the severe disease is accompanied in most cases with other organ dysfunction, frequently renal failure, but reports also indicate the association with acute respiratory distress syndrome (ARDS) and severe anemia [76, 77]. Other diseases, such as leptospirosis [78], typhoid fever [79] and viral hepatitis may occur in association with P. vivax [80] causing icteric syndromes needing to be systematically investigated in areas endemic for both diseases. As revised elsewhere [81], hyperbilirrubinemia, and clinical jaundice, may be triggered by clearance of large number of irbcs by the spleen; local liver injury due to hypoxia in cases of cythoadherence/rosetting, mostly seen in falciparum malaria and evidenced by serial elevation of transaminases AST/ALT; systemic infections and endotoxemia; disseminated intravascular coagulation (DIC) associated with microangiopathic hemolysis; intrahepatic cholestasis; drug-induced jaundice due to the use of mefloquine, artesunate/amodiaquine. Jaundice could also be caused by failure in bilirubin excretion due to the aforementioned factors [81, 82] METABOLIC COMPLICATIONS Metabolic acidosis (MA) is one of the most reported metabolic alterations in malaria. Most of what is known about MA is derived from Pf infection reports and very little is mentioned about the presence of metabolic acidosis in vivax malaria studies [56, 83, 84], probably because the phenomenon of cythoadhesion is the hallmark of Pf infection, leading to vascular occlusion and hypoxemia. MA is defined as plasma bicarbonate <15mmol/L and hyperlactatemia is defined as lactate >5mmol/L, both common in falciparum malaria and good predictors of bad outcome [85-87]. Also referred as lactic acidosis, MA has multiple etiology and is normally associated with other disturbances,

20 10 MALARIA i.e., acute respiratory distress syndrome (ARDS), multiple-organ dysfunction syndrome (MODS), severe dehydration, severe anemia, hypoglycemia, and cerebral malaria. Pathogenic mechanisms include elevation of lactic acid concentrations in blood as the result of anaerobic glycolysis due to insufficient tissue perfusion. The major mechanisms are low tissue perfusion related to hypotension and shock, microvascular occlusion by adherent and sequestered irbcs and impaired oxygen delivery to tissues due to severe anemia [88]. As little evidence of Pv-iRBs sequestration in microvasculature of vital organs is available, it is possible that less MA takes place, as suggested and reviewed elsewhere [52, 89, 90]. Hypoglycemia is defined by blood glucose < 40 mg/dl and can be accompanied by acidemia (ph<7.3) and hyperlactatemia, which increases mortality rates in falciparum malaria [85]. Hypoglycemia affects adults, but severe cases are more frequent in children, being independently associated with poor outcome and death [91-94]. Although most of what is known about this complication comes from studies in Africa and other endemic areas for falciparum malaria, a few reports of vivax-infected patients are available [56, 76, 95]. As reviewed elsewhere [88], hypoglycemia may originate from impaired glucose production, increased glucose consumption or a combination of both. Increased glucose turnover may be the most reasonable explanation for hypoglycemia in severe malaria, although more studies are needed PREGNANCY-RELATED MALARIA Evidence from malaria endemic areas, mostly from Asia-Pacific region, shows that malaria during pregnancy causes a wide range of complications both to the pregnant woman and the fetus. These major complications vary from effects on mother (severe anemia, severe malaria presentation both in P. falciparum and P. vivax infection and in rare cases, death); fetus and neonate (low birth weight - LBW, more pronounced in vivax infection than in falciparum malaria, preterm delivery, miscarriage, stillbirth and vivax-related increased mortality in the first year of life) and also congenital malaria, which may be responsible for severe illness and sepsis-like neonatal presentation [51, 60, ]. Similar findings were found in studies from the Brazilian Amazon where P. vivax has predominated since the middle 1980s. It is suggested that the burden of malarial infection in pregnant women from this population is high, with anemia being the most common complication. Other reported complications are also LBW, vaginal bleeding, amniorrhexis, fetal loss, premature delivery, hypoglycemia, hepatitis and jaundice [90, ]. Some suggested key aspects of malaria-associated pregnancy (PAM) pathogenesis are: cythoadherence/sequestration of irbcs in the placenta, which could lead to structural and hemodynamic alterations with reduced nutrient transportation to the fetus and subsequent LBW [104]; systemic and placental inflammatory response with pro-inflammatory cytokines production. This seems to play an important role in PAM and has been associated to LBW [60, 104, 105], whereas other reports do not show inflammatory reaction

21 MALARIA PATHOGENESIS 11 with no hemozoin pigment deposition in P. vivax-infected placenta but the presence of intervillous macrophages in P. falciparum-infected placentas [106] RESPIRATORY DISTRESS AND LUNG INJURY Respiratory distress is defined by oxygen saturation less than 94%, or deep breathing (acidotic breathing), or an age stratified increased rapid respiratory rate (> 32/min in adults, > 40 in children 5-14y, > 50 in children aged 2 mo to 5 y, and > 60 in babies less than 2 mo) [107]. Current definition criteria of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are based on timing, PA chest radiograph, pulmonary artery pressure (PAP) and PaO2/FiO2 ratio. Both occur acutely, with bilateral infiltrates, PAP 18mmHg with no clinical evidence of atrial hypertension and PaO2/FiO2 ratio 300 for ALI and 200 for ARDS. Respiratory distress is a well-established severe presentation in malaria caused by P. falciparum [ ]. In malaria caused by P. vivax, severe respiratory manifestations have only recently appeared in the literature and are one of the most common severe complications of malaria infection in children [4, 56, 112, 113] and adults [5, 6, 114] living in vivax endemic areas. Reports of severe pulmonary manifestations in travelers returning from endemic areas have already been described [ ]. Most of the knowledge on adult pulmonary manifestations due to vivax malaria is based on isolated case reports [2, 115, ]. Vivax malaria may lead to respiratory distress in pregnant women and cause miscarriage [127]. Interestingly, almost all cases of respiratory manifestations, lung edema and ALI/ARDS occur after the institution of antimalarial treatment, namely chloroquine [114, 118, 128, 129], but also with other antimalarials [130], and only few reports show respiratory complications before the institution of antimalarial treatment [131, 132]. Death may be preceded by severe pulmonary complications [2, 5, 6] and case-fatality rate exceeds 40% in some studies [3, 5, 6, 13]. Based on autopsy reports from vivax mono-infection malaria, major findings include heavy and edematous lungs, alveolar capillary congestion with mononuclear and neutrophilic infiltrates, and signs of diffuse alveolar damage with focal areas of hyaline membrane formation [2, 114]. Several factors are suggested as contributors to lung pathology and respiratory decline in vivax malaria, albeit precise mechanisms are poorly understood. Altered pulmonary physiology includes impaired ventilation, airflow obstruction, reduced gas transfer, and increased phagocytic activity. Sequestration of P. vivax infected red blood cells (Pv-iRBCs) in lung microvasculature and host immunological response to lung-specific sequestrated irbcs, or to systemic malaria infection seem to co-participate in the pathogenesis of malaria lung, however more information is needed regarding the role of cythoadhesion in endothelial lung cells [52] and severe disease.

22 12 MALARIA NEUROLOGICAL SYNDROME AND COMA Neurological impairment in malaria-infected patients is a high mortality-associated manifestation with varying clinical presentations. Impaired consciousness or unrousable coma (Blantyre coma scale 2 in children or Glasgow coma scale 10 in adults), prostration with generalized weakness such that the patient is unable to walk or sit up, failure to feed, and multiple convulsions (more than two episodes in less than 24h) are some of the principal clinical manifestations of neurological severity in malaria. Coma and brain injury may have distinct pathogenic mechanisms according to different sets of patients, i.e., children and adults, or different Plasmodia infection, vivax and/or falciparum. In severe falciparum malaria, coma is a frequent complication occurring in almost half of the severe patients, and it is a strong predictor of fatal outcome and occurs similarly in adults and in children [46, 133]. Coma is not frequently reported in vivax malaria patients. In a study from Indonesian Papua, coma was 23 times less frequent in vivax mono-infection than in falciparum disease [134] and in the Brazilian Amazon autopsy series, coma was reported to be present in only three of the 17 vivax-infected patients [135]. A case was recently described of a PCR-confirmed P. vivax monoinfection in a man that presented signs of partial thrombosis in cerebral computed tomography suggesting cerebral malaria. This patient had no medical history that could lead to the presented alterations [136]. Pathologic features of vivax-associated neurological syndrome are poorly characterized [51, 90] and causes other than non-vivax malaria, such as co-infection by P. falciparum and bacterial/viral infections, might participate in neurological dysfunctions leading to coma. In a pediatric series, five vivax malaria children presented coma but viral encephalitis and previous neurological sequelae were also present [56]; in the Papuan study, 7 out of 31 deceased children with falciparum malaria had coma and died from non-malarial causes and/or mixed infections [137]. Caution is needed when attributing coma uniquely to malaria. Hypoglycemia, metabolic acidosis, bacterial and viral meningoencephalitis must be investigated as additional causes [90]. Other neurological complications due to vivax malaria include tremor in a patient after recovery from coma [134], facial diplegia [138], acute demyelinating polyneuropathy [139] and blindness associated to residual weakness due to acute ischemic infarction of a branch of cerebral artery with no history that could lead to this presentation prior to vivax infection [140]. Ocular alterations are also present in malaria and assessment of retinal microvasculature has been suggested to be of good diagnostic and prognostic significance [141, 142]. Retinal hemorrhages have been reported in vivax infections with no signs of neurological alterations [143, 144] and in other studies, falciparum, but not vivax malaria, ocular manifestations are suggested to be related to cerebral malaria and bad prognosis [145, 146].

23 MALARIA PATHOGENESIS ACUTE RENAL FAILURE Patients presenting oliguria and creatinin levels above 3.0 mg/dl are defined as patients with acute kidney failure (ARF). It has been reported in both Pf and Pv severe patients [76, 77, 80, 135, 147, 148], with some needing dialysis, and some evolving to death [76, 149, 150]. Increasing numbers of children with renal impairment in severe vivax malaria has also been reported [55, 83, 151], and some studies show that renal impairment occurred similarly or more frequently in vivax malaria [152, 153]. In pediatric populations ARF seems to be less common, as well as less severe, when compared to adult populations with falciparum malaria infection [133]. In adults, as well as in children, ARF seems to be associated with multi-organ dysfunction syndrome (MODS) [55, 76, 83, 147, 153] and bacterial sepsis, dehydration, and shock, and history of chronic renal failure should be investigated [90]. An autopsy study showed that pre-existing co-morbidities were present in six cases presenting acute kidney injury [135]. Other possible findings include thrombotic microangiopathy, hemolytic uremic syndrome, and glomerulonephritis [156]. Biopsies of malarial patients with ARF show acute tubular or cortical necrosis with presence of eosinophils, thrombotic microangiopathy, characterized by endothelial injury, microvacular occlusion by platelet or fibrin thrombi and glomerular ischemia [150]. Several mechanisms are proposed to explain ARF in malaria but pathogenesis remains mostly not understood. Falciparum malaria-associated ARF is linked to endothelial injury and thrombi formation, where host immune response with cytokines and reactive oxidative species (ROS) and nitrous oxide (NO) increase vascular permeability, renal vasoconstriction, as well as restriction of kidney blood flow due to irbcs sequestration and tissue hypoxia may be implicated [157]. The presence of angiopoietins and vascular endothelial growth factor has been shown to participate in endothelial dysregulation [158] and could lead to ARF pathogenesis. In Pv infections endothelial dysfunction [42] could also participate in malaria-associated kidney injury ALGID MALARIA/SHOCK Algid malaria refers to malaria-related circulatory shock. Circulatory and hemodynamic collapse usually is defined by systolic blood pressure under 70mmHg in adults and below 50mmHg in children, as well as non-responsiveness to fluid reposition. Adult reports come from several endemic and non-endemic areas [76, 80, 159, 160], and may also be seen in the less frequent infection with P. knowlesi [162]. Shock in children is less frequent [55, 83], but a report from the Brazilian Amazon showed high frequency of shock in pediatric ICU patients irrespective of the species (56). Shock presentation usually is related to other severe manifestations becoming part of MODS, and may present in a sepsis-like shock syndrome. Bacteremia was associated with higher fatality in falciparum malaria in Africa [163].

24 14 MALARIA ATYPICAL MANIFESTATIONS Atypical malarial complications are not infrequent in the literature. Although severe rhabdomyolysis is typically reported in association to falciparum malaria [164, 165], a single case was reported in a patient with vivax malaria in the Brazilian Amazon [166]. Idiopathic thrombocytopenic purpura (ITP) is an auto-immune disease characterized by low platelet count in the absence of other causes of thrombocytopenia. A patient with vivax malaria presented ITP after receiving antimalarial therapy (chloroquine), with subsequent malarial episodes all triggering platelet count drops [167]. Splenic hematomas are rarely found, and despite being less common in falciparum [168], it can lead to splenic rupture and has a fatal outcome [169]. Lower abdominal pain should always be investigated in vivax malaria. Short-term and long-term cognitive impairment is well studied in children with severe falciparum malaria. Attention, memory, visual-spatial skills, language and executive functions are impaired in children that became sick with malaria during early infancy [ ]. A study conducted in the Brazilian Amazon, a vivax endemic area with unstable transmission, showed that non-severe malaria caused a negative impact on school performance of children [175]. Other malaria-related atypical manifestations are acalculous colescysthitis [176] and child gangrene [177]. Most of the atypical manifestations are not well understood and pathogenic mechanisms deserve more study. 1.7 COMORBIDITIES The presence of other diseases concurrent to malaria infection may contribute to severe presentation and fatal outcome. To what extent acute and chronic infectious and non-infectious diseases contribute to this is unknown. Generally, malaria endemic areas are also endemic for a wide range of infectious diseases such as HIV, leptospirosis, dengue, tuberculosis, hepatitis virus and also non-infectious diseases as emphysema, diabetes and heart failure, each of them having unique pathological aspects. In a Brazilian autopsy series of vivax malarial patients, more than 70% of the patients presented some other factor that may have contributed to malaria complication leading to worse outcomes. Some examples include: decompensated cirrhosis with associated coagulopathy in a patient that developed lung edema and severe gastrointestinal bleeding; emphysema in a patient who developed ARDS; and congestive heart failure in a patient who developed MODS [135]. In the pediatric series of children with severe malaria, also from the Brazilian Amazon, a large portion of the children had some other concomitant infection, such as gastroenteritis, pneumonia and bacterial sepsis, which probably worsened the general presentation of malaria (56). The exacerbated inflammatory response found in malaria may also worsen some adjacent infectious or non-infectious, chronic or acute disease. Recent evidence shows that bacteremia was associated to increased fatality in children with falciparum malaria [163]. Therefore, pathogenesis studies must consider ruling out co-morbidities in order to understand the mechanisms related to the parasite per se. On

25 MALARIA PATHOGENESIS 15 the other hand, understanding the pathogenesis of co-infections is also necesssary considering that they are more common than ever thought in neglected populations.