Malaria

11

Pathogenesis of Malaria Pathogenesis of Malaria Recent conceptsRecent concepts

Dr. R. Geetha M.D.Dr. R. Geetha M.D.Prof. of PathologyProf. of Pathology

GEETHAGEETHA 22

•Caused by intracellular protozoan parasite Plasmodium

•Vector – Anopheles female mosquitoes i.e. males don’t bite.

•40 % of world population is at risk

GEETHAGEETHA 33

GEETHAGEETHA 44

GEETHAGEETHA 55

Gametes

MacroMicro

GEETHAGEETHA 66

GEETHAGEETHA 77

GEETHAGEETHA 88

Pre-Erythrocytic schizonts

GEETHAGEETHA 99

Merozoites

GEETHAGEETHA 1010

•Merozoites take approx. 20 secs to invade a red cell.

•Four distinct steps are involved in this process of invasion

Presenter

Presentation Notes

Merozoites rapidly (approximately 20 seconds) and specifically enter erythrocytes. This specificity is manifested both for erythrocytes as the preferred host cell type and for a particular host species, thus implying receptor-ligand interactions. Erythrocyte invasion is a complicated process which is only partially understood at the molecular and cellular levels (Gratzer and Dluzewski 1993). Four distinct steps in the invasion process can be recognized (Figure): initial merozoite binding reorientation and erythrocyte deformation junction formation parasite entry

GEETHAGEETHA 1111

Initial merozoite bindingInitial merozoite binding

Initial interaction is by random Initial interaction is by random collisioncollisionPresence of merozoite surface Presence of merozoite surface protein has been implicated. (MSP 1)protein has been implicated. (MSP 1)MSP MSP –– 1 is capable of secreting 1 is capable of secreting proteolytic enzymesproteolytic enzymes

GEETHAGEETHA 1212

Reorientation and RBC DEFORMATIONReorientation and RBC DEFORMATION

After binding to red cell the parasite After binding to red cell the parasite reorients itself so that the apical end is reorients itself so that the apical end is juxtaposed to the cell membrane.juxtaposed to the cell membrane.There is transient red cell deformationThere is transient red cell deformationAMA AMA –– 1 antigen has been implicated in 1 antigen has been implicated in this process.this process.This antigen is localized at the apical end This antigen is localized at the apical end of the merozoiteof the merozoite

GEETHAGEETHA 1313

Role of apical organelles in red cell invasionRole of apical organelles in red cell invasion

Three distinct apical organelles have been Three distinct apical organelles have been identified in E.M. studiesidentified in E.M. studiesThese are micronemes, rhoptries and dense These are micronemes, rhoptries and dense granulesgranulesThe contents of these organelles are expelled as The contents of these organelles are expelled as the parasite invades the red cellthe parasite invades the red cellMicronemes are expelled first with initial contact Micronemes are expelled first with initial contact with the red cell membranewith the red cell membraneRhoptries are discharged nextRhoptries are discharged nextDense granules are released after the parasite Dense granules are released after the parasite has completed its entryhas completed its entry

Presenter

Presentation Notes

Specialized secretory organelles are located at the apical end of the invasive stages of apicomplexan parasites. Three morphologically distinct apical organelles are detected by electron microscopy: micronemes, rhoptries, and dense granules (Table). Dense granules are not always included with the apical organelles and probably represent a heterogeneous population of secretory vesicles. The contents of the apical organelles are expelled as the parasite invades, thus suggesting that these organelles play some role in invasion. Experiments in Toxoplasma gondii indicate that the micronemes are expelled first and occur with initial contact between the parasite and host (Carruthers and Sibley 1997). An increase in the cytoplasmic concentration of calcium is associated with microneme discharge (Carruthers and Sibley 1999), as is typical of regulated secretion in other eukaryotes. The rhoptries are discharged immediately after the micronemes and the release of their contents correlate with the formation of the parasitophorous vacuole. Dense granule contents are released after the parasite has completed its entry, and therefore, are usually implicated in the modification of the host cell. For example, RESA is localized to dense granules in merozoites and is transported to the host erythrocyte membrane shortly after merozoite invasion (Culvenor 1991). However, subtilisin-like proteases, which are implicated in the secondary proteolytic processing of MSP-1 (discussed above), have also been localized to Plasmodium dense granules (Blackman 1998, Barale 1999). If MSP-1 processing is catalyzed by these proteases, then at least some dense granules must be discharged at the time of invasion.

GEETHAGEETHA 1414

GEETHAGEETHA 1515

Junction formationJunction formation

•Junctions form between the parasite and host cell

•Microneme proteins are important in junction formation

•These are EBA-175 (erythrocyte binding antigen of falciparum), DBP (Duffy binding protein from vivax), SSP2 (sporozoite surface protein).

•These proteins bind to receptors on the red cell surface

GEETHAGEETHA 1616

Parasite EntryParasite EntryTight junction is formed between the Tight junction is formed between the parasite and the red cell membraneparasite and the red cell membraneA merozoite enzyme serine protease A merozoite enzyme serine protease cleaves the band 3 zone of Red cell cleaves the band 3 zone of Red cell membranemembraneAn incipient An incipient parasitophorousparasitophorous vacuolar vacuolar membrane formsmembrane formsThis membrane This membrane invaginatesinvaginates along with the along with the parasitic component in to the red cellparasitic component in to the red cell

GEETHAGEETHA 1717

CytoadherenceCytoadherenceParasite modifies surface Parasite modifies surface of red cell to enhance of red cell to enhance survival.survival.Modifications cause Modifications cause adherence of red cells to adherence of red cells to the endothelium the endothelium Knob protrusions appear Knob protrusions appear on the surface of infected on the surface of infected red cellred cellThis is also known as This is also known as sequestrationsequestration

Presenter

Presentation Notes

The parasite modifies the surface of red cells. These modifications cause adherence of red cell to the endothelium causing rosette formation. Knob protrusions appear on the surface of red cells causing an area of contact between the cell surface and the endothelium.

GEETHAGEETHA 1818

•They secrete proteins that form 100 – nm bumps on the red cell surface , called knobs or sequestrins.

•These are encoded by var genes, so called because they exhibit antigenic variations.

•These sequestrins bind to endothelial cells by ICAM -1, the thrombospondin receptor, and the glycophorin CD46.

Presenter

Presentation Notes

Red cells containing immature ring forms of the parasite, which are flexible can pass through spleen, circulate in the blood, where as red cells containing mature schizonts, which are more rigid avoid seqestration in the spleen. In addition, sequestrin causes red cells to bind to and form rosettes with uninfected red cells. Cerebral involvement by P.falciparum which causes 80% of deaths in children is due to adhesion of the P. falciparum parasites to endothelial cells within the brain. Patients with cerebral malaria have increased amounts of ICAM – 1, thrombospondin receptor, and CD 46 on their endothelial cells within the brain

GEETHAGEETHA 1919

Advantages of sequestrationAdvantages of sequestration•The major advantage is the avoidance of spleen thus preventing removal of diseased red cells

•Low oxygen tension in deeper tissues may provide better environment for the survival and metabolism of the parasite

•This feature could also help the parasite in eluding the immune mechanism of the host

•Contributes to higher reproductive capacity

GEETHAGEETHA 2020

Presenter

Presentation Notes

A schematic model depicting some possible mediators of cerebral malaria. The cytoadherence of infected erythrocytes to brain endothelial cells (BEC) and the release of exo-antigens could stimulate the BEC and immune effector cells such a macrophages (MF) to secrete cytokines. These cytokines, such as tumor necrosis factor-a (TNF), would lead to an increased expression of possible endothelial cell receptors (eg., ICAM-1) and promote an increase cytoadherence of infected erythrocytes. Large numbers of bound infected erythrocytes could lead to vascular blockage and hypoxia and have localized metabolic effects (eg., hypoglycemia, lactic acidosis). The increased number of infected erythrocytes and exo-antigens would also lead to higher cytokine levels. TNF-a is also known to stimulate nitric oxide (NO). Nitric oxide can affect neuronal function by interferring with neurotransmission. Nitric oxide also causes vasodilation which could lead to the intracranial hypertension associated with cerebral malaria

GEETHAGEETHA 2121

Young ring form trophozoites

GEETHAGEETHA 2222

GEETHAGEETHA 2323

GEETHAGEETHA 2424

GEETHAGEETHA 2525

ImmunityImmunityInnate resistance (common in endemic Innate resistance (common in endemic areas)areas)Specific immunity against merozoitesSpecific immunity against merozoitesRole of liver and spleenRole of liver and spleenThese are not sterilizing immunityThese are not sterilizing immunity

GEETHAGEETHA 2626

Innate resistanceInnate resistance

Genetically determinedGenetically determinedDuffy negativeDuffy negativeOvalcytosisOvalcytosis (Band 3 zone of red cell (Band 3 zone of red cell membrane is disordered)membrane is disordered)Sickle cell anaemiaSickle cell anaemiaThalassemiaThalassemiaG6PD deficiencyG6PD deficiency

Presenter

Presentation Notes

Certain genetic diseases and polymorphisms have been associated with decrease infection or disease (Box). For example, individuals which lack the Duffy blood-group antigen are refractory to P. vivax. A large proportion of the populations in western Africa are Duffy negative, thus accounting for the low levels of P. vivax in west Africa. This innate resistance led to the identification of the Duffy antigen as the erythrocyte receptor for merozoite invasion. (See receptor-ligand interactions during merozoite invasion.) Several inherited erythrocyte disorders are found predominantly in malaria endemic areas and at frequencies much higher than expected. This has lead to speculation that these disorders confer some protection against malaria. For example, southeast Asian ovalcytosis is due to a mutation in an erythrocyte membrane protein called band 3. This mutation causes the erythrocyte membrane to become more rigid and more refractory to merozoite invasion. The mechanism(s) by which the other diseases might confer protection against malaria are not known. In most cases it is presumed or speculated that the combination of the defect and infection leads to premature lysis or clearance of the infected erythrocyte. For example, glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes would have an impaired ability to handle oxidative stress (see Drug Action). The additional oxidants produced as a result of parasite metabolism and the digestion of hemoglobin (see Biochemistry Notes) may overwhelm the infected erythrocyte and lead to its destruction before the parasite is able to complete schizogony.

GEETHAGEETHA 2727

AnemiaAnemia

Repeated Repeated hemolysishemolysis of red cellsof red cellsImmune & Non immune Immune & Non immune hemolysishemolysis of non of non infected red cellsinfected red cellsIncreased Increased splenicsplenic clearanceclearanceReduction of red cell survivalReduction of red cell survivalDyserythropoiesisDyserythropoiesis in bone marrowin bone marrowDrug induced Drug induced hemolysishemolysisNORMOCYTIC NORMOCHROMIC ANAEMIANORMOCYTIC NORMOCHROMIC ANAEMIA

GEETHAGEETHA 2828

Bone marrowBone marrow

DyserythropoiesisDyserythropoiesisIron sequestrationIron sequestrationErythrophagocytosisErythrophagocytosisThese are common in the acute These are common in the acute phasephase

GEETHAGEETHA 2929

LiverLiver

HepatomegalyHepatomegalyBrown/gray/black in colorBrown/gray/black in colorMalarial hepatitisMalarial hepatitis

GEETHAGEETHA 3030

Hepatic PhaseHepatic Phase

•Malarial sporozoites having single surface antigen invade liver cells within minutes of infection.

•The single surface antigen makes it a target for vaccines.

•HLA-B53 associated resistance to P. falciparum in certain African groups is due to their ability to prevent this hepatic stage damage.

Presenter

Presentation Notes

Malarial sporozoites, the stage transmitted by mosquito bites, have a single antigen on their surface which is an important vaccine candidate. Sporozoites released into the blood stream within minutes attach to and invade liver cells by binding to the hepatocyte receptors for thrombospondin and properidin located in the baso lateral layer. These two are serum proteins. Within liver cells these parasites multiply rapidly. In fact as many as 30,000 merozoites ( which are asexual and haploid forms) are released when the hepatocyte ruptures. These merozoites are notorious for changing their surface antigen constantly thereby defeating the immune mechanism of the body. The HLA-B53 associated resistance to P. falciparum exhibited by many Africans appears to be caused by the ability of HLA – B53 to present livercell stage specific malarial antigens to cytotoxic T cells, which then kill malaria infested hepatocytes.

GEETHAGEETHA 3131

The liver tissue at autopsy exhibits stasis of red cells containing coarse malaria pigment in the sinusoid, and activation of Kupffer cells, phagocytizing the infected red cells (Giemsa). In case of falciparum malaria, the red cells infected by trophozoites and schizonts are trapped to the endothelial cells via CD36 and ICAM-I, so that these cells are not seen in the peripheral blood.

GEETHAGEETHA 3232

At autopsy, the spleen is soft, swollen and black in color (gross findings). Histologically, the red pulp is filled with erythrocytes having concentrated malaria pigments.

GEETHAGEETHA 3333

spleen, showing numerous schizonts and schizont- phagocytizing macrophages (Giemsa).

GEETHAGEETHA 3434

SpleenSpleen

SplenomegalySplenomegalyFibrotic spleenFibrotic spleenSplenicSplenic ruptureruptureTropical Tropical splenomegalysplenomegaly

GEETHAGEETHA 3535

CNSCNS

Involved only in falciparum infectionsInvolved only in falciparum infectionsBrain is edematousBrain is edematousMalarial Malarial granulomagranulomaPunctatePunctate haemorrhageshaemorrhagesMalarial encephalitis and Malarial encephalitis and meningoencephalitismeningoencephalitisDurcksDurcks granulomagranuloma

GEETHAGEETHA 3636

Pathophysiology of cerebral malariaPathophysiology of cerebral malaria

Caused by falciparum malariaCaused by falciparum malariaSequestration plays an important roleSequestration plays an important roleThere is mechanical blockage to cerebral There is mechanical blockage to cerebral circulation due to circulation due to cytoadherencecytoadherenceHost mediators like cytokines also play an Host mediators like cytokines also play an important role in the pathogenesisimportant role in the pathogenesis

GEETHAGEETHA 3737

GEETHAGEETHA 3838

Increased morbidity and mortality of falciparum malaria is due to

• Release of large number of merozoites

• All erythrocytes are invaded

• Sequestration causing immune evasion and

complications

Presenter

Presentation Notes

P. falciparum can produce serious disease with mortal consequences. This increased morbidity and motality is due in part to the high parasitemias associated with P. falciparum infections. The parasitemia can also rapidly increase due to the sequestration of P. falciparum, which minimizes removal of infected erythrocytes by the spleen, and the lack of preference for a particular subclass of erythrocytes (eg, reticulocytes or senescent; see species differences). The high parasitemia and sequestration result in other complications associated with falciparum malaria, the most notable being anemia and cerebral malaria. The anemia is due in part to the destruction of erythrocytes during blood-stage schizogony. Furthermore, non-infected erythrocytes are destroyed at higher rates during the infection and there is a decreased production of erythrocytes. Severe anemia probably accounts for most of the falciparum malaria associated deaths among children in sub-Saharan Africa.

GEETHAGEETHA 3939

KidneyKidney

Acute diffuse malarial nephritisAcute diffuse malarial nephritisNephroticNephrotic syndromesyndromeAcute renal failureAcute renal failure

GEETHAGEETHA 4040

GEETHAGEETHA 4141

LungLung

Acute pulmonary oedemaAcute pulmonary oedemaLobar pneumoniaLobar pneumoniaBronchopneumoniaBronchopneumonia

GEETHAGEETHA 4242

CVSCVS

Functional abnormalitiesFunctional abnormalitiesMicrocirculatory changes in coronary Microcirculatory changes in coronary vesselsvessels

GEETHAGEETHA 4343

GITGIT

Ischemia of gutIschemia of gutNecrosisNecrosisUlcerationUlceration

GEETHAGEETHA 4444

PathogenesisPathogenesisRupture of infected red cellsRupture of infected red cellsRelease of parasite materials and Release of parasite materials and metabolites metabolites haemazoinhaemazoin and cellular debrisand cellular debrisIncreased activity of Increased activity of reticuloreticulo endothelial endothelial system i.e. system i.e. hepatosplenomegalyhepatosplenomegaly

GEETHAGEETHA 4545

DiagnosisDiagnosis

Microscopic testsMicroscopic testsNonNon--microscopic testsmicroscopic testsRapid diagnostic testsRapid diagnostic tests

GEETHAGEETHA 4646

Microscopic testsMicroscopic tests

Involves staining and direct Involves staining and direct visualisation under microscopevisualisation under microscopeThese are peripheral smear study These are peripheral smear study and Quantitative buffy coat test and Quantitative buffy coat test (QBC)(QBC)Stains used are Stains used are GiemsaGiemsa, , LieshmannLieshmann, , RomnowskyRomnowsky and and JaswanthJaswanth singhsinghBattacharyaBattacharya

GEETHAGEETHA 4747

Erythrocytic PhaseErythrocytic Phase

•Merozoites bind to the surface of Red blood cells.

•They release proteases from specific organelle called rhoptry.

•They multiply rupture and invades other red cells.

•Some of them develop into sexual forms i.e. gametes.

•These gametes infect the mosquitoes when they suck blood.

Presenter

Presentation Notes

Once released the merozoites bind by a parasite lectin-like molecule to sialic residues on glycophorin molecules on the surface of red cells. In fact P.vivax merozoites bind by a homologous lectin to the Duffy antigens on the red cells. Many Africans who are DUFFY NEGATIVE ARE RESISTANT TO THIS PARASITE.. The merozoites release multiple proteases from a special organelle called the rhoptry. Within the red cells the parasites multiple in a membrane bound digestive vacuole, hydrolyzing hemoglobin through secreted enzymes that include an aspartate protease . Individuals with sickle cell trait are resistant to malaria because their red cells sickle when parasitized and so are removed by the spleen. Most malarial parasites within the red cells develop into merozoites, rupture the cell, and then infect new red cells, some parasites develop into sexual forms called gametocytes that infect the mosquito when it takes its blood meal.

GEETHAGEETHA 4848

Differences between plasmodium Differences between plasmodium speciesspecies

FalciparumFalciparum VivaxVivax OvaleOvale MalariaeMalariae

Numerous Numerous rings, smallerrings, smaller

Enlarged red Enlarged red cellcell

Similar to Similar to vivaxvivax

Compact Compact parasiteparasite

No No trophozoites/ trophozoites/ schizontsschizonts

SchuffnerSchuffner’’ss dotsdots

Compact Compact trophozoitetrophozoite, , elongated red elongated red cellcell

CresentCresent shaped shaped gametocytesgametocytes

Amoeboid Amoeboid trophozoitetrophozoite

Fewer Fewer merozoitesmerozoites

Merozoites Merozoites form rosette form rosette formationformation

GEETHAGEETHA 4949

Presenter

Presentation Notes

The ring forms of all four species are very similar and difficult to distinguish. P. falciparum rings tend to be a little smaller and more numerous than the other species. The presence of a large number of rings in the absence of more mature stages, as well as multiply-infected erythrocytes, is highly suggestive of P. falciparum. Erythrocytes infected with P. vivax and P. ovale are enlarged and exhibit Schüffner's dots as the rings mature into trophozoites. The trophozoites of P. vivax are often ameboid, whereas P. ovale tends to be more compact. The P. malariae trophozoite is very compact and the host erythrocyte is not enlarged. Mature asexual forms of P. 2 falciparum are rarely found in the peripheral circulation. The typical number of merozoites produced per schizont is: P. vivax 14-20 (up to 24), P. ovale 6-12 (up to 18), P. malariae 8-10 (up to 12), and P. falciparum 16-24 (up to 36). P. falciparum exhibits crescent-shaped gametocytes whereas the other species are all round to oval. P. vivax and P. ovale gametocytes are in enlarged erythrocytes with Schüffner's dots and are difficult to distinguish from each other. P. malariae gametocytes do not modify the host erythrocyte. Gametocytes can be distinguished from trophozoites by their large size (nearly filling the erythrocyte) and a single nucleus. Mature microgametocytes tend to stain lighter than macrogametocytes and have a more diffuse nucleus.

GEETHAGEETHA 5050

Peripheral smear studyPeripheral smear study

This is a gold standard testThis is a gold standard testThick and thin smears are preparedThick and thin smears are preparedThick smears are to identify the Thick smears are to identify the parasitesparasitesThin smears to identify the speciesThin smears to identify the speciesRomanowskyRomanowsky stain is usedstain is used

GEETHAGEETHA 5151

Thick film Thick film

A number of stains like Field’s, Giemsa’s, Wright’s and Leishman’s are suitable for staining the smears. Thick films are ideally stained by the rapid Field’s technique or Giemsa’s stain for screening of parasites. The sensitivity of a thick blood film is 20 parasites/µl (0.0004%) parasitaemia. Thin blood films stained by Giemsa’s or Leishman’s stain are useful for specification of parasites and for the stippling of infected red cells and have a sensitivity of 200 parasites/µl (0.004%). The optimal pH of the stain is 7.2

GEETHAGEETHA 5252

Thin film Thin film

An experienced technician can detect as few as 5 parasites/µl in a thick film and 200/µl in a thin film.

Health & Medicine

Malaria