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Why is falciparum malaria so severe?

1. High parasitemia Large number of merozoites per

schizont (30,000 merozoites) Severe anemia

2. Will infect any stage of RBC No preference

3. Sequestration Only ring and early trophozoites

are found in circulating blood Immune evasion - avoids

clearance by the spleen 3. Cytoadherence

Cerebral malaria

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2

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Pathogenesis of falciparum Malaria

Main problem - Cytoadherence Induction of cytokines (rupture of

RBCs) leads to an increase in thelevel of endothelial adhesionmolecules

Vascular occlusions Infected RBCs will adhere to

endothelial cells as well as to eachother (rosetting, clumping) Rosetting was found in 50% of field

isolates - correlated well with severityof disease Rosetting Clumping

Result of Cytoadherencecytoadherence

↓cerebral ischemia

(Cerebral Malaria - ~10%of hospital cases)

↓hypoxia, metabolic

effects↓

coma↓

deathCerebral Malaria accounts for

80% of deaths

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Knobs and cytoadherence Knobs

Electron denseprotuberance found onRBC membrane ofinfected cells

Composed of parasitederived proteins that areexported to RBC surface

F: ring stageProtein is in parasite

G,H: young trophozoitesProtein is found in RBC cytoplasm

I: mature trophozoitesProtein is nearly completely localized

to the RBC membrane surface

What mediates cytoadherence?

Proteins synthesized by the parasite are transported to theRBC membrane

KAHRP, PfEMP2 remain at the subsurface of RBC membrane Possibly reorganize submembrane cytoskeleton to form knobs

PfEMP1 transmembrane protein - exposed on the RBC surface One player that mediates adherence to endothelial cells Cytoplasmic domain (acidic) interacts with KAHRP (Highly basic)

RBC

Parasite and PV not pictured

Plasmodium Cytoadherance

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PfEMP-1 Structure

Cloned and sequenced Family of 40-50 var genes (variant)

Conserved intracellular ATS Acidic terminal segment - binds KAHRP

Transmembrane domain - tm Conserved head structure

Cysteine-rich interdomain region Duffy binding like domain

Variable extracellular domain 2-7 copies of DBL

Antigenic switching is accompanied bychanges in binding phenotype

Possible Host CellReceptors

• CD36• Ig super-family

• ICAM-1• VCAM-1• PECAM-1

• E-selectin• thrombospondin• chondroitin sulfate A• hyaluronic acid

• Rosetting Receptors• CR-1• glycosaminoglycan• blood group A

Very big topic - look for review on antigenic variation

Summary

Only P. falciparum infections result in cytoadherence Knob production

Parasite modifies host RBC via exported proteins Tubovesicular network

PfEMP-1 may be the main mediator of cytoadherence PfEMP-1 undergoes antigenic variation

Family of var genes Recombination mediated at telomeric or subtelomeric ends during

meiosis

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Pathogenesis of malaria

Parasitemia

Cerebral malariaAnemia

Immunity to Malaria High endemic areas

People do develop immunity - slow to develop, short-lived Not sterilizing immunity - does not protect against infection Premunition - immunity that is dependent upon parasite being present

Mainly resistance to a superinfection Tolerant or asymptomatic carriers - parasitemias are present

Sporozoite infection

Can be asymptomatic

Clinicalsymptoms

Innate Resistance

Duffy blood group antigens Fy/Fy - no merozoite binding

Sickle cell anemia Mutation is hemoglobin (single aa sub.) Homozygous individual suffer greatly from

disease - life expectancy ~30 years Heterozygous individual are protected

Presence of sickle cell trait confers about 85-90%protection

Selective pressure has led to maintenance of adisease trait

1 in 10 African Americans are heterozygous

Thalassemia

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Malaria in the US?

All patients lived within 10 miles of Palm BeachInternational Airport

Cases presented between July 22-August 26, 2003Patient 1 and 2 attended the same party of July 4

Think about sporulation in mosquito (~10 days)Think about EE cycle (8 days) and E cycle (11 days)

First reported outbreak of Malaria with extended transmission in US since 1986

Malaria Treatments

Drugs•Antifolates

Drugs•Quinolines interact withHaem/maemozoin

Drugs•Atovoquone inhibition of CytC reductase

Drugs•Antibiotic inhibiting protein translationSuch as tetracycline

Malaria Treatments Quinine related drugs

Quinine Chloroquine Mefloquine Halofantrine

Antifolates Sulphadoxine Pyrimethamine Proguanil

Other drugs Artemisinin Clindamycin Atovoquone

Why chloroquine?

Synthesized in 1934 First used in WWII Most widely used drug Safe, fairly cheap to make

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Cinchona the source of quinine

Peruvian Indians seem tobe the first to know aboutthe potential benefits ofquinine as they chewedbark while working in coldstreams and mine for theSpanish

In the early 1600s the barkwas used to treat the feverof the Countess ofChinchon and became wellknown as Jesuit’s powder orPeruvian bark

Cinchona the source of quinine Not until 1820 was quinine isolated -

was also used for flavoring in tonicwater - gin and tonics were used forprophylaxis of malaria

Initial preparations were often quitevariable in the amount of activeingredient resulting in varyingeffects

Other reasons to mistrust the drugwere the fact that it was first usedby Jesuits (Oliver Cromwell e.g.died from malaria due to refusal totake the “powder of the devil)

A little history on cinchona High demand had brought the Cinchona tree almost to

extinction in the wild

Charles Ledger a trader in Peru sent out Manuel IncraMacrami to locate a stand of special tree they had foundearlier

After three years Manuel came back with 15 kg of seedswhich they sold for 100 guilders to the Dutch, but theBritish were not interested

C. ledgeriana formed the basis of a very profitable Dutchquinine monopoly which lasted until World War II

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Chloroquine - an alternative

Chloroquine an artificial quinineanalog was a very potent drugwhich was very cheap to makeand had no really serious sideeffects

Acts within 24-48 hrs

None of the drugs developedsince come close to chloroquine

Hemoglobin degradation Proposed chloroquine mode of action

- still debated Weak base, uncharged @ pH7 Rapid diffusion through plasma and

lysosomal membranes Inside lysosome - charged molecule is

trapped Accumulation via conc. Gradient Chloroquine binds FP - interferes with

pigment formation Inhibits heme polymerase

HIGHLY TOXIC TO PARASITE May also interfere with nulceic acid

biosynthesis

digestive vacuole is site of Hb digestionseveral proteases required (plasmepsinsand cysteine proteases) endpoint ofdigestion is a toxic heme productferriprotoporphyrin (FP)FP is polymerized to form hemozoin, the“malaria pigment” which is thought todetoxify heme for the parasite

Drug resistance

Ability of a pathogen to survive and/or multiply despite theadministration and absorption of the drug given in doses equal toor higher than doses normally given.

Important factors associated with resistance:

Longer half-life Single mutation for resistance Poor compliance - use chloroquine to treat any fever - takes 2-

3 doses in stead of 10 Host immunity Large numbers of people using the drug

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Chloroquine resistance Characterized using the following:

Sensitive (S): asexual parasite count reducesto 25% of the pre-treatment level in 48 hoursafter starting the treatment and completeclearance after 7 days, without subsequentrecrudescence - Complete Recovery.

RI, Delayed Recrudescence: asexualparasitemia reduces to < 25% of pre-treatmentlevel in 48 hours, but reappears between 2-4weeks.

RI, Early Recrudescence: asexual parasitemiareduces to < 25% of pre-treatment level in 48hours, but reappears earlier.

RII Resistance: Marked reduction in asexualparasitemia (decrease >25% but <75%) in 48hours, without complete clearance in 7 days.

RIII Resistance: Minimal reduction in asexualparasitemia, (decrease <25%) or an increasein parasitemia after 48 hours.

Spread of chloroquine resistance

1960

1989

1978

1965

Spread of chloroquine resistance

Used since the early 1950s Effective and safe treatment

Widespread use of chloroquine First cases were seen in the

late 1950s at two different foci Columbia Cambodia-Thailand border

Interesting transfer to theAfrican continent - spread veryquickly at that point

Now additional resistance Mefloquine antifolates

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Combined Resistance

CDC, 2005

Mechanisms of drug resistance

Changes in target enzyme Mutations in target gene Decreased affinity to drug

Overexpression of target Amplification

Decreased activation of drug

Changes in accessibility less import, or more export of drug

Resistance to chloroquine

Drug resistant parasites showlower chloroquineconcentrations in the foodvacuole - decreasedaccumulation

This seems to be due to anincreased pH of this acidicorganelle (chloroquine is aweak base)

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Resistance to chloroquine

In vitro data suggests that aseries of point mutations in thenewly identified membranetransporter PfCRT areresponsible for resistance

This putative transporter localizesto the membrane of the foodvacuole

Large field studies have foundstrong association of thesemutations with chloroquineresistance (different mechanismin P. vivax)

PfCRT, resistance mutations highlighted

Malaria antifolates While Plasmodium parasites cannot

synthesize purines de novo, they dosynthesize pyrimidines de novo.

Folates - important cofactors in thisprocess (also for amino acidbiosynthesis) and are made de novo aswell - Plasmodium cannot utilizepreformed folates.

Pyrimethamine and sulfa drugs(Fansidar) act synergistically as theytarget the same pathway at two differentpoints

Humans take up folate as a vitamin inthe food.

Malaria antifolates Pyrimethamine and

proguanil are dihydrofolateanalogs inhibiting parasiteDHFR but not humanDFHR

Leads to depletion of folatepool and subsequentlyreduces the pool ofthymidine for nucleic acidsynthesis

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Mutations Associated withDrug Resistance

Drug Major MutationsGene

Sulfadoxine +pyrimethamine

Sulfadoxine +pyrimethamine

Chloroquine

Mefloquine

Chloroquine

Dihydropterote synthase

Dihydrofolate reductase

Chloroquine resistance transporter

Multidrug resistance gene

Multidrug resistance gene

A437G, K540E, A581G

S108N, N51I, C59R, I164L

K76T

D86Y

Increased copy number

Prevention of drug resistance Drug selection based on type of case

Avoid drugs with longer-half lives if possible

Do not use anti-malarials for non-malarial indications Use of chloroquine to treat rheumatoid arthritis is endemic area

Ensure patient compliance with treatment

Monitoring for resistance and early treatment

Clear policies for use of new anti-malarials

Use drugs in combination to inhibit emergence of resistance Drugs that target two different biological pathways

Artemisinin - New Treatment? An ancient herbal remedy with good

potency! (Qinghaosu) Earliest document use of herbal

remedy dating back to 168 BC. Extract from the wormwood plant Great attention since 1998 as a “new”

treatment for malaria Can be used for cases that exhibit

multi-drug resistance Extremely fast acting - within 12 hr Inhibits the trophozoite stage

Artemisia annua

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Artemisinin - Current Status Cultivation and extraction: takes up to

9 months Artemisinin and its derivatives can cure

malaria within 3 days Drug stability issues - light sensitive 2001: WHO recommended Artemisinin-

based combination therapy (ACT) Backing from company Novartis to

supply at cost 2004: projected 10 Million doses needed 2005: projected 60 Million doses needed

Chemical synthesis Jay Keasling - Scientist of the Year

(2006) http://keaslinglab.lbl.gov/wiki/index.php/Main_Page

HUGE SHORTAGE

Supply and demand drove the cost up

Atovaquone

HIGHLY Lipophilic compound -essentially insoluble in water

Resembles Ubiquinone

Alone - fast recrudescence in patientsdecreased parasite susceptibilityfollowing treatment(High relapse rate!)

Ubiquinone

Atovaquone

Atovaquone

Interferes with parasite mitochondrialelectron transport - collapses themembrane potential

Also effective against Toxoplasma andPneumocystis

Plasmodium parasites developedresistance to atovaquone quickly Mutations in cyt bc1 complex

Combined therapy: Malarone Atovaquone + proguanil

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Prophylaxis - Malarone

Atovaquone/Proguanil