Molecular markers for the detection of malaria drug resistance

Presented by-

Anil kumarM.Sc PHE Ist yearVCRC(ICMR)

NEED…???

• As per 2015, 214 million new cases reported for malaria worldwide(African region-88%, South East Asian region-10% and East Mediterranean region-2% ).

• About 438,000 deaths worldwide.• 3.2 billion people (about half of the population of world)

live in high risk zone of malaria.• About 12 billon USD per year expenses on malaria

(illness, treatment and diagnosis) globally.• In 2014 itself 535 death in India. • Drug resistance increases the burden of disease,

mortality and financial expenses.

INTRODUCTION• Malaria is caused by mosquito vector.• Parasites involved are - P. falciparum, P. malariae, P. vivax and

P. ovale.• Different drugs are known for different mechanism of action.• 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, but within the limits of tolerance of the patients is known as drug resistance.

• Resistance from drug of choice is because of different mechanisms associated with respective drugs.

• First anti malarial drug resistance started in 1957 in South East Asia region against chloroquinine.

DISEASE BURDEN….

DISTRIBUTION OF DRUG RESISTANT MALARIA

Drug Introduction “First” year resistance reported

Difference (years)

Quinine 1632 1910 278

Chloroquine 1945 1957 12

Proguanil 1948 1949 1

Sulfadoxine-pyrimethamine

1967 1967 <1

Mefloquine 1977 1982 5

Atovaquone 1996 1996 <1

Artemisinins 1971 1998 35

Development of drug resistance

REASONS FOR DEVELOPING RESISTANCE..??

Unregulated or poorly administered antimalarial drug use.

Combination drug therapy use without a complementary drug.

The malaria parasite increases its capacity to repair the damage

caused by anti malarial drug gives it a higher chance of survival.

Because the drug is more effective against the parasite at its later

stage of development, the parasite slowed down its growth so it

could survive longer in the younger stages.

Long residual effect of antimalarial drugs.

DRUGS OF CHOICE…..Combination therapy:

•Mefloquine+Artesunate•Sulfadoxine/Pyrimethamine+Artesunate•Lumefantrine+Artemether

Single-Agent Therapy:

•Chloroquine•Amodiaquine•Slfadoxine/Pyrimethamine•Mefloquine•Halofantrine•Quinine•Tetrayclin/Doxycyclin•Atovaquone•Primaquine•Artemisine compounds

MECHANISM OF DRUG ACTION&

RESISTANCE DEVELOPMENT

Chloroquine, Amodiaquine, Quinine, Quinidine, Mefloquine,

Primaquine Lumefantrine and

Halofantrine.

•Theses drugs kills the erythrocytic stage of

parasite.•Primaquine also kills the intrahepatic forms

and gametocytic stages.

QUINOLINE DERIVATIVE

Choloroquine :Mode of action- Accumulating in plasmodium food vacuole leading to inhibition of utilization of heme and producing complexes which are toxic to plasmodium.Resistance-Among Chloroquine resistant parasites, there is a decrease in accumulation of drug within the food vacuole.Molecular basis-•Mutation in the gene encoding the “Chloroquine Resistance Transporter(Pf CRT)” in chromosome 7 (alteration K76T). •In SEA S163R mutation(fourth transmembrane domain) was found along with K76T.

Amodiaquine :It is similar to chloroquin and available in coformulation with artesunate and treated in chloroquine resistance cases.Side effects agranulocytosis and hepatotoxicity.Molecular basis for resistance-Pfcrt and Pfmdr1

Quinine and QuinidineMode of action-

Depresses oxygen uptake and

carbohydrate metabolism; disrupting

Na/K channel, intercalates into DNA, disrupting parasites

replication and transcription.

Resistance –Gene associated pfcrt1,

pfmdr1 and pfnhe1

4-methanolquinolines

Quinoline - methanol compound

Affective against all asexual stages of different malarial

parasites.Mode of action similar to

CQ

Resistance-Gene associated pfmdr1

Mefloquine

Similar to 4-methanolquinolines in structure. Used against

chloroquine & Mefloquine resistant infections.

Mode of action same as CQ.

Resistance –Gene associated pfcrt1 &

pfmdr1

Lumefantrine & halofantrine

Mode of action- It is a competitive inhibitor

of ubiquinol, specially inhibiting the mitochondrial

ETS at the bc1 complex. Inhbition of bc1 activity

results in a loss of mitochondrial function.

Resistance-Single nucleotide

polymorphism in the cytochrome b gene

Atovaquone

Drugs include: Pyrimethamine,

Sulphonamides, Proguanil and Dapsone.

Mode of action –Drug synergistically target enzymes involved in Folate

synthesis (DHFRase).Resistance –

Due to mutational changes in the plasmodium DHFRase enzyme.

Resistance can be delayed by using in combination

with Sulphonamide

Antifolates

Mode of action – Sulfadoxine interferes with

bacterial folic acid synthesis and growth via competitive inhibition of para-aminiobenzoic acid; pyrimethamine inhibits microbial dihydrofolate reductase, resulting in

inhibition of tetrahydrofolic acid synthesis

Resistance-

Sulfadoxine – Pyrimethamine

Point mutations in pfdhfr and pfdhps associated genes.

Mode of action –It act by binding iron, breaking down peroxide bridges leading

to the generation of free radicals that damage parasite

proteins.They act rapidly by clearly all blood stages and gametocytes

of plasmodium species.

Resistance :

Pf kelch protein gene on chromosome 13 (kelch13)

Artemisinin

Drug Mutations Chloroquine pfcrt (K76T) codons:72-76; pfmdr1 (P. falciparum multidrug

resistance transporter 1) codon: 86Y/Y184/1246Y Quinine pfcrt, pfmdr1, and pfnhe1 (P. falciparum sodium/proton

exchanger 1)Amodiaquine pfcrt, pfmdr1

Mefloquine Increased copy number of pfmdr1

Piperaquine Pfcrt :76T; pfmdr 1 codon: 86Y/Y184/1246Y

Lumefantrine Pfcrt codon: 72-76; pfmdr1 N86/184F/D1246 or increased copy number

Atovaquone Single nucleotide polymorphisms in the cytochrome b gene

SP Point mutations in pfdhfr and pfdhpsArtemisinin Pf kelch protein gene on chromosome 13 (kelch13)

Sulfadoxine pfdhps: 437G/540E

Pyrimethamine pfdhfr : 51I/59R/108N- triplet mutation

P. vivax PvMDR1, pvdhfr and pvdhps

Genes involved in drug resistance

Response to therapy

EVALUATION OF DRUG RESISTANCEENDEMIC REGION:

EPIDEMIC REGION:PCR

REFERENCES

Articles:

•Ingrid B Müller and John E Hyde ., Antimalarial Drugs: Modes of Action and Mechanisms of Parasite Resistance; Future Microbiol. 2010;5(12)•Carol Hopkins Sibley., Understanding drug resistance in malaria parasites: Basic science for public health; Molecular & Biochemical Parasitology 195 (2014) 107–114 •Hiasindh Ashmi Antony and Subhash Chandra Parija., Antimalarial drug resistance: An overview; Trop Parasitol. 2016 JanJun; 6(1): 30–41•Liwang Cui aand et al., Antimalarial Drug Resistance: Literature Review and Activities and Findings of the ICEMR Network; Am. J. Trop. Med. Hyg., 93(Suppl 3), 2015, pp. 57–68

WWW :

•www.malariasite.com/drug-resistance/

THANKSTHANKS