The document discusses drug resistance against malaria parasites. It covers the life cycle of malaria parasites, symptoms of malaria, common areas where malaria occurs, and how malaria is transmitted. It then discusses various antimalarial drugs used for treatment, including mechanisms of action and resistance. Resistance occurs through mutations in genes encoding for drug targets in the parasite or efflux pumps. Maintaining compliance with combination drug therapies can help delay the emergence of resistance.

1. DRUG RESISTANCE
AGAINST MALARIA

2. MALARIA
Malaria is caused by a parasite called Plasmodium, which is transmitted via
the bites of infected mosquitoes.
In the human body, the parasites multiply in the liver, and then infect red
blood cells. Usually, people get malaria by being bitten by an infective female
Anopheles mosquito.
Only Anopheles mosquitoes can transmit malaria and they must have been
infected through a previous blood meal taken on an infected person.
About 1 week later, when the mosquito takes its next blood meal, these
parasites mix with the mosquito’s saliva and are injected into the person
being bitten.
When a mosquito bites an infected person, a small amount of blood is taken
in which contains microscopic malaria parasites
Malaria can also be transmitted through blood transfusion, organ transplant,
or the shared use of needles or syringes contaminated with blood.

3. Life cycle

4. INFECTIOUS OR UNINFECTIOUS
 Malaria is not infectious it can only be passed on by parasites. When
the mosquito bites you it will take some blood. If the mosquito has
the plasmodium parasite in it, the blood from its last meal, will get
infected. The next person it bites will receive the infected blood and
infect them with malaria.
 Malaria is not infectious it can only be passed on by parasites. When
the mosquito bites you it will take some blood. If the mosquito has
the plasmodium parasite in it, the blood from its last meal, will get
infected. The next person it bites will receive the infected blood and
infect them with malaria

5. WHERE DOES MALARIA COMMONLY OCCUR
 Malaria commonly occurs in Africa, Asia, South America, Central
America, Southern Mexico, Caribbean, Europe and North America. The
most common place it occurs is Sub-Saharan Africa. They have many
Plasmodium falciparum's which is the most dangerous species, of four,
that causes malaria. Plasmodium falciparum is a parasite.

6. MALARIA SYMPTOMS

7. MALARIA SYMPTOMS
 Symptoms of malaria may include fever, chills, vomiting, diarrhea,
cough, stomach, pain and muscular aches and weakness.
 If infected with the malaria parasite, Plasmodium results in the most
severe form of malaria and if left untreated, it can cause serous
illnesses. Like seizures, mental confusion, kidney failure, coma and
death

8. WHAT CAUSES MALARIA
 Humans develop malaria when infected with one of the four
protozoans from Plasmodium. A protozoan is a single celled organism.
Plasmodium is a scientific name for a parasite.

9. IS MALARIA SPREAD BY VECTORS
 Malaria is spread by vectors. A vector is a carrier In malaria mosquito
serves as the vector that carries and transfers the infectious agent
(Plasmodium), injecting it with a bite.

10. Treatment
Many classes of drugs have either important or ancillary effects on one
or more stages of the life cycle of different species of the malaria
parasites. Those presently available include the following:
 cinchona alkaloids (quinine,quinidine)
 4aminoquinolines (chloroquine,amodiaquine)
 8aminoquinolines (primaquine)
 diaminopyrimidines (pyrimethamine)
 sulfonamides and sulfones (sulfadoxine,dapsone)
 quinoline methanols (mefloquine)
 tetracyclines (tetracycline,doxycycline)
 biguanides (proguanil)
 phenanthrene methanols (halofantrine)
 hydroxynaphthoquinones (atovaquone)

11. RESISTENCE
 Drug resistance is the ability of the parasite species to survive and/or
multiply despite the administration and absorption of a drug given in
doses equal to or higher than those usually recommended but within the
limit of tolerance.
 Ability of a parasite strain to survive and/or to multiply despite the
administration and absorption of a drug given in doses equal or higher
than those usually recommended
 The emergence of resistance in Plasmodium depends on multiple
 factors, including
(i) the mutation rate of the parasite
(ii) the fitness costs associated with the resistance mutations
(iii) the overall parasite load
(iv) the strength of drug selection
(v) the treatment compliance.

12. The important factors that are
associated with resistance are
 Longer half-life.
 Single mutation for resistance.
 Poor compliance
 Host immunity.
 Number of people using these drugs.
 Drug resistance is most commonly seen in P. falciparum.
 Only sporadic cases of resistance have been reported in vivax malaria.
 Resistance to chloroquine is most prevalent

13. Degree of resistance
 WHO has developed a simple scheme for estimating the degree of
resistance that involves studying the parasitemia over 28 days.
 Smears on day 2, 7 and 28 are done to grade the resistance as R1 to
R3. In a case of normal response parasite count to fall to 25% of pre-
treatment value by 48 hours and smear should be negative by 7 days.

14. RI, Delayed Recrudescence
 The asexual parasitemia reduces to < 25% of pre-treatment level in 48
hours, but reappears between 2-4 weeks.

15. RI, Early Recrudescence
 The asexual parasitemia reduces to < 25% of pre-treatment level in 48
hours, but reappears earlier.

16. RII Resistance
 Marked reduction in asexual parasitemia (decrease >25% but <75%) in
48 hours, without complete clearance in 7 days.

17. RIII Resistance
 Minimal reduction in asexual parasitemia, (decrease <25%) or an
increase in parasitemia after 48 hours.

18. This classification however has some limitations
1. In endemic areas it is not easy to differentiate recrudescence from re-
infection.
2. Recrudescence can occur beyond 28 days also.
3. Therapeutic failure could be due to other causes also.
4. RII is a very broad category.
5. Practical difficulties in following the patient for 28 days.
6. Intermittent nature of parasitemia in the blood

19. Prevention of drug resistance
 Resistance develops most rapidly when a population of parasite
encounters subtherapeutic concentration of antimalarial drugs.

20. The following points will be helpful in
reducing the emergence of resistance:
 Selection of drugs - Use conventional drugs first in uncomplicated
cases. Greater the exposure, higher will be the emergence of
resistance.
 Avoid drugs with longer half-life if possible
 Ensure compliance
 Avoid basic antimalarials for non-malarial indications (e.g.
Chloroquine for rheumatoid arthritis in a malarial endemic area).
 Monitoring for resistance and early treatment of these cases to
prevent their spread.
 Clear policy of using newer antimalarials.
 Use of combinations to inhibit emergence of resistance

21. Mechanism of Action of Antimalarial Drugs
and resistance:
Quinine:
 The drug is a potent blood schizonticide against all four species of human
malarial parasites, it is an
 arylamino alcohol, The molecular mechanism by which quinine acts
against P. falciparum is only
 partially understood. Similar to chloroquine, quinine has been
demonstrated to accumulate in the
 parasite’s digestive vacuole (DV) and can inhibit the detoxification of
heme, an essential process within
 the parasite
 Recent studies show that the genetic basis for resistance to quinine is
complex, with multiple genes

22. Mechanism of Action of Antimalarial Drugs
and resistance:
Quinine: cont.
 influencing susceptibility. Currently, three genes have been associated
with altered quinine response:
 pfcrt (P. falciparum chloroquine resistance transporter),
 pfmdr1 (P. falciparum multidrug resistance transporter 1), and
 pfnhe1 (P. falciparum sodium/proton exchanger 1),
 all of them encoding for transporter proteins

23. Chloroquine:
 Chloroquine is a 4aminoquinoline, the principal target is the heme detoxification
pathway in the DV,
 where the parasite degrades erythrocytic hemoglobin and polymerizes the
liberated toxic heme
 monomers to inert biocrystals of hemozoin, As it accumulates in the DV,
chloroquine binds to hematin,
 a heme dimer [30]. This interaction prevents the detoxification of free heme,
leading to the buildup of
 heme monomers that permeabilize the membrane, resulting in the eventual
death of the parasite
 Polymorphisms in PfCRT have been demonstrated to be the main chloroquine
resistance determinant.
 In some parasite strains PfMDR1 can also modulate the degree of chloroquine
resistance, indicatingthat some alleles and overexpression of PfMDR1 may
increase the concentration of chloroquine within
 the DV by active transport.

24. Amodiaquine:
 Amodiaquine, also a 4aminoquinoline, the antimalarial activity is thought
to be exerted by the primary
 metabolite, monodesethylamodiaquine, which has a halflife of 9–18 days.
Based on structural
 similarity, amodiaquine is hypothesized to act by inhibiting heme
detoxification, and has been shown to
 accumulate within the DV and to bind to heme in vitro, Crossresistance
between chloroquine and
 amodiaquine has been reported and mutations in PfCRT and PfMDR1 are
associated with decreased
 susceptibility to both drugs. However, crossresistance is incomplete and
some chloroquine resistant
 parasites remain susceptible to amodiaquine.

25. Mefloquine:
 Mefloquine is a 4methanolquinoline, Although the exact mechanism of action
remains unclear, in vitro
 experiments demonstrate that mefloquine can bind to heme and exert some
antimalarial activity by
 inhibiting heme detoxification,it has been shown that mefloquine inhibits the
import of other solutes
 into the DV and might therefore also target the PfMDR1 transport function
itself, Resistance to
 mefloquine is mediated by amplification of pfmdr, leading to overexpression
of this resident DV
 membrane transporter, studies on transgenic parasites expressing different
pfmdr1 copy numbers,
 observed a reduced parasite susceptibility to mefloquine with increased
PfMDR1mediated import into
 the DV

26. Piperaquine:
 Is 4aminoquinoline piperaquine, studies have shown that piperaquine
accumulates in the DV and that
 it is a potent inhibitor of heme polymerization, Modulation of piperaquine
susceptibility by mutations
 in PfCRT have been confirmed
Lumefantrine:
 It is structurally related to the hydrophobic arylamino alcohol antimalarials,
Polymorphisms in
 PfMDR1, particularly the variant N86, and amplification of the encoding gene
(pfmdr1) have been
 associated with reduced susceptibility to lumefantrine in Africa and Asia.

27. Primaquine:
 Primaquine is an 8aminoquinoline, Several studies have suggested that
primaquine binds to PfCRT
 and can thereby inhibit chloroquine transport, possibly leading to a synergistic
action between the two
 antimalarials and a reversal of chloroquine resistance, Primaquine is
contraindicated in patients with
 certain subclasses of glucose6phosphate dehydrogenase (G6PD, encoded on the
X chromosome)
 deficiency, due to the risk of a severe reaction resulting in hemolytic anemia

28. Atovaquone:
Atovaquone is a lipophilic hydroxynaphthoquinone analog structurally
related to ubiquinol (an
 important coenzyme in the electron transport chain within the
mitochondria) and is used for treatment
 of apicomplexan parasites, including Plasmodium, Toxoplasma,
Theileria and Babesia, Molecular
 evidence exists that atovaquone specifically targets the cytochrome
bc1 complex, located in the inner
 mitochondrial membrane, thereby inhibiting the respiratory chain,
resistance is conferred by single
 nucleotide polymorphisms in the cytochrome b gene.

29. Antifolate drugs:
 Antifolate drugs:
 The antifolate drugs used for malaria therapy are the sulfa drugs
sulfadoxine and dapsone that inhibit
 the dihydropteroate synthetase enzyme (PfDHPS), and pyrimethamine
and proguanil, which inhibit the
 dihydrofolate reductase (PfDHFR) activity of the bifunctional
dihydrofolate reductase/thymidylate
 synthase enzyme. In addition to PfDHFR, proguanil may target other
pathways, Unfortunately,
 resistance due to point mutations in both target enzymes emerged
quickly after introduction, it is now.

30. Antifolate drugs:
 primarily used as intermittent preventative malaria treatment during
pregnancy and, to a lesser extent
 for the treatment of malaria infection(due to high prevalence of
parasites resistant to the drug
 combination in endemic regions), Unfortunately dapsone caused
hemolysis in G6PD deficient patients,
 and therefore this combination therapy is no longer recommended.

31. Artemisinins:
 Artemisinins have a unique trioxane structure with an endoperoxide
bond that is required for
 antimalarial activity, The mechanism of action of artemisinin drugs is
not fully understood, but the
 prevailing theory is that the endoperoxide bridge is cleaved, leading
to the formation of reactive carbon
 radicals that subsequently alkylate essential biomolecules.
 Mutations in pfatp6 (P. falciparum Ca2+ transporting ATPase 6) have
been associated with decreased
 artemether susceptibility and polymorphisms in ubp1, encoding for a
deubiquitination enzyme, are
 associated with increased artesunate resistance in the rodent malaria
parasite Plasmodium chabaudi

32. Drug resistance resistance mechanisms:
Chloroquine resistance:
 As the malaria parasite digests haemoglobin, large amounts of a toxic
by-product are formed. The parasite polymerizes this by-product in its
food vacuole, producing non-toxic haemozoin (malaria pigment), It is
believed that resistance of P. falciparum to chloroquine is related to
an increased capacity for the parasite to expel chloroquine at a rate
that does not allow chloroquine to reach levels required for inhibition
of haem polymerization, This chloroquine efflux occurs at a rate of
40 to 50 times faster among resistant parasites than sensitive ones

33. Antifolate combination drugs:
Antifolate combination drugs, such as sulfadoxine + pyrimethamine, act
through sequential and synergistic blockade of 2 key enzymes involved with
folate synthesis. Pyrimethamine and related compounds inhibit the step
mediated by dihydrofolate reductase (DHFR) while sulfones and sulfonamides
inhibit the step mediated by dihydropteroate synthase (DHPS). Specific gene
mutations encoding for resistance to both DHPS and DHFR have been
identified. Specific combinations of these mutations have been associated
with varying degrees of resistance to antifolate combination drugs.
Atovaquone:
Atovaquone acts through inhibition of electron transport at the
cytochrome bc1 complex. Although resistance to atovaquone develops
very rapidly when used alone, when combined with a second drug, such as
proguanil (the combination used in MalaroneTM) or tetracycline,
resistance develops more slowly. Resistance is conferred by single-point
mutations in the cytochrome-b gene.

34. Molecular approach to Antimalarial drug
resistance:
PfCRT:
Intensive chloroquine chemotherapy has led to the emergence of resistant P.
falciparum strains, Analysis of the progeny from a genetic cross between the
chloroquine sensitive strain and a chloroquine resistant strain identified a 36 kb
region on chromosome 7 responsible for chloroquine resistance, Subsequent
examination of the locus identified the P. falciparum chloroquine resistance
transporter gene (pfcrt), encoding a transmembrane protein of 424 amino acids
and localized within the DV membrane, Homologs of PfCRT have been identified
in several Plasmodia species (P. vivax, Plasmodium yoelii, P. chabaudi, P. knowlesi,
Plasmodium berghei) and CRT-like proteins exist in non-related organisms such as
Cryptosporidium parvum, Dictyostelium discoideum, and Arabidopsis thaliana
Several biochemical studies of the parasitic DV, comparing parasites expressing a
mutant or a wild type pfcrt allele, demonstrated that chloroquine accumulates
within the DV and that parasites with mutant PfCRT accumulate less chloroquine
than parasites expressing wild type PfCRT. The most plausible explanation for this
difference in accumulation is that chloroquine resistant parasites can export
chloroquine via active transport.

35. PfMDR1:
 The P. falciparum multidrug resistance transporter 1, The gene, present on
chromosome 5, encodes an ATP-binding cassette (ABC) protein of 1419 amino
acid, It has been demonstrated that PfMDR1 resides, like PfCRT, within the
membrane of the DV.

 The endogenous function of MDR-like proteins in other organisms consists of
the translocation of a variety of substrates including sugars, amino acids,
peptides, proteins, metals, inorganic ions, toxins and antibiotics across
cellular membranes, Mutations in MDR transporters in mammalian cancer
cells lead to a decreased intracellular drug accumulation, increased drug
efflux, and cross resistance to structurally unrelated drugs. The MDR
phenotype is often mediated by gene amplification, resulting in
overexpression of the protein


36. PfMDR1:
 PfMDR1 has been shown to import the fluorophore Fluo-4 into the parasitic
DV, mediated by a functional ATP-binding cassette. Therefore, PfMDR1 might
act as a general importer, functioning to sequester toxic metabolites and
drugs into the DV. It might also indirectly influence drug flux by affecting
intracellular ion gradients, such as chloride ions, or pH

 From analysis of field isolates, five amino acid positions (86, 184, 1034, 1042
and 1246) have been reported to influence susceptibilities to lumefantrine,
artemisinin, quinine, mefloquine, halofantrine and chloroquine,
amplification of PfMDR1 is associated with reduced susceptibility to
lumefantrine, artemisinin, quinine, mefloquine, and halofantrine and
deamplification of PfMDR1 leads to an increase in chloroquine resistance

37. PfMRP:
The multidrug resistance-associated protein (PfMRP) belongs, like
PfMDR1, to the family of ATP-binding cassette (ABC) proteins. More
specifically, it belongs to the ABC transporter C subfamily, Pfmrp,
located on chromosome 1, encodes an 1822 amino acid protein of,
which localizes to the plasma membrane and membrane-bound vesicles
within the parasite in asexual and sexual erythrocytic stages, The
protein is not essential in the blood stages, but genetic disruption leads
to increased parasite susceptibility to several antimalarial drugs like
chloroquine, quinine, artemisinin, piperaquine and primaquine and
accumulates more GSH, chloroquine and quinine . Additionally, the lack
of expression of PfMRP leads to a fitness cost of the parasite in in vitro
culture at parasitemias above 5%, which might be due to a an impaired
transport of toxic compounds out of the parasite. There is some
evidence from linkage studies based on in vitro drug susceptibility
assays with field isolates that point mutations (Y191H and A437S) reduce
susceptibility to chloroquine and quinine

38. PfNHE:
The P. falciparum Na+/H+ exchanger (pfnhe) is a candidate gene within a locus
on chromosome 13, it is a 1920 amino acids protein, and is predicted to have
12 transmembrane domains and to be localized to the parasitic plasma
membrane, it is speculated that it actively effluxes protons to maintain a pH of
7.4 within the parasite, countering acidification by anaerobic glycolysis, the
parasite’s main source of energy.
PfNHE contains three microsatellite regions and the increase of DNNND repeat
number in microsatellite ms4670 has been associated with decreased quinine
susceptibility but not in others.

39. Folate pathway:
The folate pathway provides the parasite with cofactors that are essential
for the production of pyrimidines for DNA replication and the metabolism of
several amino acids. Two enzymes, dihydropteroate synthase (PfDHPS) and
dihydrofolate reductase activity of the bifunctional enzyme, dihydrofolate
reductase-thymidylate synthase (PfDHFR-TS) are currently targeted by
antimalarial drugs.
PfDHPS is involved in producing a folate precursor and is inhibited by the
sulfur-based drugs sulfadoxine and dapsone. PfDHFR is responsible for
reducing dihydrofolate into tetrahydrofolate and its function can be
impaired by the action of the antifolate drugs pyrimethamine and
cycloguanil, the bio-activated metabolite of proguanil, Resistance has
emerged in the late 1980s, and is now wide-spread with point mutations in
both pfdhfr and pfdhps implicated in resistance.

40. Cytochrome bc1 complex:
The cytochrome bc1 complex catalyzes the transfer of electrons from
ubiquinol to cytochrome c, which is coupled to the translocation of protons
across the inner mitochondrial membrane, thereby maintaining the
membrane potential of mitochondria used to produce ATP by an ATP
synthase, The antimalarial drug atovaquone can inhibit the parasitic
cytochrome bc1 complex by causing a collapse in the mitochondrial
membrane potential, which is lethal for the parasite, Several mutations
within the cytochrome b gene can lead to atovaquone resistance, with
most mutations altering the ubiquinol binding site of the protein
Artemisinin resistance:
Genetically stable and transmissible artemisinin (ART) and artesunate (ATN)
resistant malaria parasites has been selected in the rodent malaria parasite
Plasmodium chabaudi, Resistant parasites have mutations in PfATP6, a Ca++
ATPase and putative drug target.

41. Geographical distribution of antimalarial
drug resistance

42. References: