why is falciparum malaria so severe? · cerebral malaria 1 2 3 pathogenesis of falciparum malaria...
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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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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