Malria: diagnosis, treatment and prevention

Diagnosis
1. Microscopy
  1. thin or thick blood films
  2. Can tell four species apart
2. Antigen
  1. Cannot distinguish between all types of malaria
  2. Serves as a rapid diagnostic tool
3. PCR
  1. Expensive
Treatment
1. Quinine-based drugs
  1. Act within the food vacuole
  2. Quinine
    1. 1st treatment available for malaria
      1. Natural medicine
        Peruvian mexicans chewed the bark of the Cinchona plant to treat malaria
        Earkly 1600's it was used by the catholic missionaries (called it "Jesuit's powder")
        Purified in 1817 - Pelletier and Caventou
      2. First pharmaceutical agent = 4-methanol quinolone
        Could cause cinchonism
        Blurred vision, nausea, vomitting, imparied hearing and dizziness
        Active against RBC stage malaria
        Effective against chloroquine-resistant malaria
    2. Found in tonic water
    3. Pharmacokinetics
      1. Absorption
        Oral
      2. Distribution
        70% is bound to protein
      3. Elimination
        Redily metabolised in liver (~80%)
        Metabolites inactive
        20% in urine
        Half life of ~18hrs
  3. Chloroquine
    1. 4-aminoquninolone
      1. Artificial analogue of quinine
        Developed in 1940's
    2. Cheap, stable and no serious side effects
    3. Active against RBC stages
    4. Used extensively in 60's and 70's (during erradication campaign)
      1. Lead to massive levels of resistance
        e.g. in Vietnam etc.
    5. No other anti-malarial has come close
    6. Pharmacokinetcs
      1. Absorption
        Oral
      2. Distribution
        50-60% protein bound
      3. Elimination
        Partially metabolised in liver in to active de-ethylated metabolites
        Excreted in urine unchanged (45%) - but slowly
        Half life = 1-2 months
  4. MOA of Qunine and Chloroquine
    1. e.g. during ring-stage
      1. Parasite trophozoite requires haemoglobin (Hb) to survive
        Hb taken up by cytosome (endocytosis) uptake -> transport vesicles - > fuse with food vacuole
        Hb in food vacuole
        Hb broken down to release amino acids (released into cytoplasm) and haematin (kept in food vacuole)
        AA's used by cell
        Haematin (toxic to parasite) is crystallised to form haemozoin (inert, non-reactive)
        Two haematin molecules dimerised with a propionate group of one haematin interacting with the Fe ion of a second (this de-toxfies the Fe)
        These dimers form crystalline structures with other dimers -> haemozoin
        Seen as the second solid structure during the ring phase (the other one is the nucleus)
        Dimer = 4-beta haematin
        Fe(3+) ion of haematin can generate free radicals
    2. Quinine and chloroquine accumulate in the food vacuole
      1. Prevent the formation of haemozoin crystals -> haematin builds up, generates free radicals and kills the parasite
        In the 60's chloroquine was heralded as a major success
        However chloroquine resistance was observed as early as 1959 in SE Asia and S. America
        Since then chloroquine resistance has spread to all endemic areas (1957 - spread through asia and oceana, 1959-60 spread through S. America)
        In 1978 resistance from Asia spread throughout sub saharan africa
        Resistance now common
        Molecular basis for resistance
        chloeroquine resistance develops slowly
        Spectrum (low-high) resistance seen
        Suggestions mechanism of resistance is complex
        Resistance at target level (unlikely)
        Target is haematin (synthesised by host - Hb)
        Cannot be altered by parasite)
        Resistance at drug level (likely)
        Less chloroqunine retained in parasite
        Either; reduced uptake or drug efflux
        In low-medium resistant strains
        Mutations detected (by PCR and sequencing) in parasite protein PfCRT (P. falciparum chloroquine resistance transporter)
        PfCRT is found in the membrane of the food vacuole
        Features 10 transmembrane domains
        Resistance-conferring mutatin (K76T) is localised in a region of the protein involved in substrate selectivity
        Mutation: K76T is a key diagnostic tool for resistance detection
        In non-resistant strains food-vacuolar chloroquine is positively charged (protonated) due to the low pH
        The lysine (K) residue at position 76 features a positive side chain which physically repels the chloroquine molecule - preventing its escape)
        Hence its accumulation to x20,000 the level of the plasma chloroquine concentration
        The mutation of K to threonine (T) at position 76 means that chloroquine is removed from the food vacuole as threonine has an uncharged side chain
        Chloroquine is eliminated from the food vacuole via PfCRT (efflux pump)
        In higher resistance strains
        Feature additional mutaiotns to K76T in PfCRT
        Resistance can be enhanced by a mutation in a second gene PfMDR (P.falciparum multi druig resistance)
        PfMDR is an ABC (ATP-binding casette) transporter - group of membrane transporter proteins
        MDRs in other organisms function to export hydrphobic drugs and indirectly regulate ionic gradients
        MDRs are responsible for drug resistance in cancer cells
        The Ca(2+) channel blocker verapamil (which reverses resistance of mammalian cells to anti-cancer cells) reverses chloroquine resistance
        PfMDR contains 12 transmembrane domains (TMDs)
        ATP interacting loops
        PfMDR (like the PfCRT) is located in the membrane of the food vacuole
        There are several known mutations unique to different geographical resistant strains
        E.g. N86Y (Africa); S1034C, N1042D and D1246Y (S. America)
        ALL RESISTANT STRAINS FEATURE K76T!!!
      2. The acidity of the food vacuole causes chloroquine to accumulate (due to protination -> become charged) to x20,000 the plasma [chloroquine]
  5. Mefloquine
    1. Used in cases of chloroquine-resistant malaria
    2. Absorption
      1. Oral
    3. Distribution
      1. Can cross the BBB (cerebral malaria)
    4. Side effects
      1. Experienced in 1:10,000 patients (psychological effects, seizures, motor and CNS problems
    5. MOA is unknonw
      1. In food vacuole like other quinines?
    6. Resistance newly emerging (SE Asia) - different mechanism to chloroquine
      1. Molecular basis of mefloquine resistance
        Requires wild type PfMDR1
        Mutation in PfMDR actually causes mefloquine sensitivity
        Mutation associated with overexpression of wild type PfMDR
        Can remove mefloquine faster from the food vacuole
        So mefloquine resistance occurs by a different method to chloroquine resistance
        Amplification of PfMDR (mefloquine)
        Mutataion in PfMDR (chloroquine)
2. Non-quinolone-based drugs
  1. Sulfadoxine-pyrimethamine combinational therapy
    1. Second-line treatment in chloroquine resistance (after mefloquine)
    2. Active against the asexual cycle merozoite -> trophozoite -> merozoite
    3. Side effects
      1. (Rare) death from drug-induce dermatological conditions (toxic epidermal necrolysis; or Steven-Johnson syndrome (SJS)
      2. Many milder side effects: rash, photosensitivity, blood disorders (aplastic anaeamia, agranulocytosis), liver/lung damage
    4. Absorption
      1. Single oral dose (3 tablets for adults)
    5. Distribution
      1. 90% is protein-bound
      2. both cross the placental barrier and pass into the breast milk
      3. Pyrimethamine concentrates in blood cells (red and white) and crosses into the CNS fluids
    6. Elimination
      1. <5% of each drug is metabolised
      2. both excreted in urine (long half lives - both >100 hrs)
    7. MOA
      1. Targets multiple enzymes of the folate synthesis pathway
        Drugs act synergistically (complimentary)
        Reduce folate levels -> folate is essential for nucleotide (DNA) synthesis and metabolism of certain amino acids
        Humans lack components of this pathway and therefore rely on the diet for folate
        Sulfadoxine
        Type 1 anti-folate
        para-aminobenzioc acid (PABA) analogue
        Necessary in folate synthesis
        Target: Dihydropteroate synthase (DHPS) - missing in humans
        Competitive inhibition
        In Plasmodium, DHPS is part of a bifunctional enzyme with dihydroptero pyrophosphokinase = PPPK-DHPS
        Pyrimethamine
        Type 2 anti-folate
        Pyrimidine containing compound
        Target: Dihydrofolate reductase (DHFR) - present in humans
        Competitive inhibition
        Pyrimethamine binds x7,000 more strongly to DHFR than dihydro folate (natural substrate)
        In Plasmodium DHFR is in copmplex with another enzyme thymidylate synthase (TS)
    8. Resistance
      1. Basis of resistance due to difference in activity (pyrimethamine is more active than sulfadoxine)
      2. Due to point mutations in their respective enzymes
        Firstly in DHFR-TS (only DHFR domain affected)
        Then in PPPK-DHPS (only DHPS domain affected)
        After four mutations in DHFR (above) - A437G; and K540E cause sulfadoxine resistance
        Diagnostic mutation = S108N (always present in resistant strains)
        causes 100 fold increase in resistance
        Secondary mutations cause increased resistance (e.g. N51I; C59R; I164L)
  2. Atovaquone
    1. Part of a combinational therapy (with proguanil - another Type 2 antifolate inhibitor [targets DHFR])
    2. Active against liver and RBC stages
      1. Casual prophylaxis (taken 1 day before travel)
    3. Pharmacokinetics
      1. Absorption
        Oral (standard malarone tablet)
      2. Distribution
        99% binds to serum albumin (extremely lipophilic)
      3. Elimination
        Not metabolised (slowly excreted in faeces, little in urine - half life = 48-72hrs)
      4. Side effects
        Very few
    4. Trade name = Malarone
    5. MOA
      1. Analogue of ubiquinone
        Ubiquinone: shuttles electrons from complexes 1 and 2 of the oxidative phosphorylation pathway to complex 3 (cytochrome b)
        Atovaquone inhibts the passing of electrons to (reduction of) complex 3 in the ETC
        Inhibits proton gradient production -> ATP production
    6. Resistance
      1. Can only arise through a point mutation (Y268N or Y268S) in the gene encoding cytochrome b
        Resistance is rare when using Malarone combinational therapy - first case reported in 2002
        Tyrosine (Y) is a bulky hydrophilic amino acid that interacts with the hydrophobic atovaquone
        Substitution to a less bulky asparagine (N) or serine (S) causes a loss of drug binding ability
  3. Doxycycline
    1. Absorption
      1. Oral
    2. Distribution
      1. 90% drug in plasma
    3. Elimination
      1. Not metabolised
      2. Excreted in urine/faeces
      3. Half life = 18hrs
    4. MOA
      1. Site of action is the apicoplast (prokaryote remenant that resembles a chloroplast - non-photosynthetic)
      2. Inhibits cell growth by inhibiting translation in the apicoplast
        Stops cell growth - doesnt kill
    5. Broad spectrum antibiotic
    6. Used as prophylaxis
    7. Active against asexual cycle
    8. Resistance not yet reported
  4. Artemisinin
    1. From leaves of Artemesia annua
      1. Problem with supply
        Not enough plant material can be grown for demmand
    2. Made from a series of isoprene units with a peroxide bond
    3. Pharmacokinetics poorly understood (oral bioavailability is poor)
      1. Synthetic compounds (Artesunsate) produced with increased H2O solubility - can be injected
    4. Active against ring stage
      1. Used against MDR malaria
        Evidence of resistance in Cambodia already
        The way forward?
        Artemisinin combinational therapy with...
        Amodiaquine
        Active against all forms of malaria (falciparum, vivax, ovale, malariae)
        Active against chloroquine resistant strains
        >20 million cases of malaria treated as of (2009)
        ...Mefloquine
        Reduced mefloquine side effects
        WHO advise use for uncomplicated falciparum infection
        Not cosidered suitable for first line treatment in African malaria
    5. MOA
      1. Uknown whether one or multiple drug targets(?)
        Inhibiton of food vacuole cysteine protease activity
        Damage to parasite's ETC in the mitochondria
        Irreversible inhibition with an ATPase (PfATP6) that pumps Ca(2+) from the cytoplasm into the ER
3. Three points of malarial chemotherapy
  1. stage to target
    1. Sporozoite
    2. Liver schizont
    3. Merozoite
    4. Trophozoite (ring stage)
    5. RBC schizont
  2. In host
    1. Liver
    2. Blood
  3. Species
    1. P. falciparum
    2. P. vivax
    3. P. ovale
    4. P. malariae
4. Anatomy of the infected RBC
  1. 1. RBC
    2. 1. Parasite (cytoplasm)
        Site of action for sulfadoxine-pyrimethamine (combinational therapy)
      2. Digestive (food) vacuole
        The food vacuole is the site of action for quinine-base drugs
      3. Nucleus
      4. Mitochondrion
        Atovaquone targets the mitochonrion
      5. Apicoplast
        Apicoplast: target for doxycycline
      6. Cytosome uptake of haemoglobin
Prevention
1. Insecticide sprays - control of adult and larval stages
  1. Particularly around breeding grounds (still water)
    1. Ecological considerations
2. Mosquito nets
  1. Contain insecticide
    1. Cheap and insecticide is "contained"
3. Prophylactic treatment
4. Control of mosquito population (introduction of sterile males)
5. Drainage and removal of breeding grounds
  1. Ecological considerations

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Malria: diagnosis, treatment and prevention

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