Molecular Markers for Failure of SulfadoxinePyrimethamine Treatment of Plasmodium falciparum and P.

1
Molecular Markers for Failure of
Sulfadoxine-Pyrimethamine Treatment of Plasmodium
falciparum and P. vivax Malaria

• Zimmerman Lab
• Melanie V. Prarat
• Case Western Reserve University
• 14 July 2004

2
Mendel and Peas

• By observing several generations of plants,
  Mendel discovered that certain traits show up in
  offspring plants without any blending of parent
  characteristics
• Three important conclusions
• Inheritance of each trait is determined by genes
  that are passed on to descendents unchanged
• An individual inherits one gene for each trait
  from each parent
• A trait may not show up in an individual but can
  be passed on to the next generation

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Genetics

• Plasmodium parasites undergo a normal Mendelian
  pattern of inheritance of genetically determined
  characters
• Blood forms are haploid
• Resistance to drugs is due to gene mutations
• Recombination between genes determining
  characters, like drug sensitivity, occurs readily
  after cross-fertilization between clones
• Multi-strain infections provide the principal
  mechanism for generating parasites with different
  recombinations of genotypes

4
Back to the Basics

• Review
• Plasmodium has a complex life cycle
• asexual multiplication and gametocyte development
  occur in the vertebrate host
• fertilization between gametes takes place in the
  mosquito vector

5
What Walliker has to say

• Genetic recombination occurs at a high frequency,
  resulting in the production of parasites with new
  genotypes
• 3D7 x HB3
• ?
• 90 recombinants
• Study showed that two genetically distinct clones
  of falciparum can undergo mating during mosquito
  transmission
• Pyrimethamine was given as the anti-malarial
  treatment ? The recombinant progeny with the HB3
  enzyme and antigen characteristics survived

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A Brief History of Fansidar

• Sulphadoxine-pyrimethamine (SP) was introduced in
  1967 as a synergistic anti-malarial drug, with
  reports of resistance occurring within a few
  years
• SP (Fansidar) is used as a second-line drug in
  areas where chloroquine is still used
• In areas that are chloroquine resistant, SP has
  become the first-line antimalarial
• There are many hyperendemic and holoendemic areas
  that are now SP resistant

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Drug-resistance worldwide
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Classification of Drug Sensitivity

• Used to assess antimalarial susceptibility
• High percentages of RII/RIII responses to SP in
  endemic areas have been documented as early as
  1994 in Tanzania
• Also found in SE Asia, southern China, the
  Amazon Basin
• High frequencies of RI/RII responses have been
  reported on the Pacific coast of South America,
  southern Asia east of Iran, and throughout
  sub-Saharan Africa and Oceania
• Resistance in Thailand started in the early 1970s
• High-level resistance to P.vivax has also been
  documented in Indonesia and New Guinea, and more
  recently, in Central America

9
Another Walliker study

• Showed through a genetic cross that pyrimethamine
  drug resistance is linked to a point mutation to
  Asn-108 in the DHFR-TS gene
• RFLP analysis showed that there was a HinfI
  fragment of 4.3kb in the 3D7 parent, and a 4.2kb
  fragment in the HB3 parents
• All pyrimethamine-resistant progeny inherited the
  HB3 RFLP, and all sensitive progeny inherited
  that 3D7 RFLP
• Identified two other point mutations that confer
  higher levels of resistance

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Dihydrofolate Reductase

• DHFR is a small enzyme that plays an essential
  role in DNA synthesis
• Catalyzes the conversion of dihydrofolate to
  tetrahydrofolate, a cofactor required for the
  biosynthesis of thymidylate, pyrimidine
  nucleotides, methionine, and glycine

DHFR-TS
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Pyrimethamine

• Pyrimethamine is one of many antifolates used as
  an antimalarial drug
• Analog of dihydrofolate similar to one of the
  rings of DHFR
• Inhibits one step of the folic acid synthetic
  pathway, preventing synthesis of pyrimidines
• Leads to inhibition of DNA synthesis

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Folic Acid Pathway
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Mechanisms of Resistance

• Many theories
• Amplification of the DHFR-TS gene
• Overexpression of DHFR
• Modification of transport of various substrates
  involved in folate biosynthesis
• Drug efflux
• P. vivax inherent resistance to antifolates due
  to conformation of the active site cavity
• Point mutations resulting in the substitution of
  amino acid residues that form the active site of
  the DHFR-TS enzyme lead to decreased affinity
  between antifolates and the enzyme

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DHFR Polymorphisms in P. falciparum

• Mutations in DHFR confer resistance to
  pyrimethamine and the other DHFR inhibitors
• Ser?Asn 108 causes moderate pyrimethamine
  resistance in vitro, with higher level resistance
  resulting from Asn?Ile 51 and/or Cys?Arg 59, and
  the addition of Ile?Leu 164 causes the highest
  level of resistance.
• DHFR Ser?Asn 108 is used as a marker for SP
  resistance because it is selected for by SP
  treatment

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P. falciparum Polymorphisms

• The substitution of asparagine or threonine at
  residue 108 reduces the drugs affinity without
  affecting the enzymes operation on its natural
  substrate
• Multiple mutations diminish the efficacy of the
  enzyme on the dihydrofolate
• Suggests that multiple mutations are unfavorable
  to the parasites in the absence of drug pressure

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DHFR Polymorphisms in P. vivax

• I13L
• Wild Type 5 MEDLSDVFDIYAICACCKVA 3
• Mutant 5 MEDLSDVFDIYALCACCKVA 3
• F57L, S58R
• Wild Type 5 SVDMKYFSSVTTYVDESKYE 3
• Mutant 5 SVDMKYFRSVTTYVDESKYE 3 (Burma)
• Mutant 5 SVDMKYLRSVTTYVDESKYE 3
  (Thailand)
• S117N/T
• Wild Type 5 VVMGRSSWESIPKQYKPLPN 3
• Mutant 5 VVMGRSNWESIPKQYKPLPN 3 (Burma)
• Mutant 5 VVMGRSTWESIPKQYKPLPN 3 (Thailand)

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P. vivax Polymorphisms

• Mutations in P. vivax dhfr codons 58 and 117 are
  considered equivalent to P. falciparum dhfr
  residues 59 and 108, respectively
• Mutations at 117 in vivax and 108 in falciparum
  arise first under drug pressure
• Highly mutated genes carry the Ser?Thr 117
  mutation
• 5 AGC AGC TGG 3 (WT)
• 5 AGC AAC TGG 3
• 5 AGC ACC TGG 3 ? highly mutated

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P. falciparum vs. P. vivax

• P. vivax 5 TTC AGG TCG 3
• P. falciparum 5 TTT CGT GCA 3
• P. vivax S58R
• P. falciparum C59R
• P. vivax 5 AGC ACC TGG 3
• P. vivax 5 AGC AAC TGG 3
• P. falciparum 5 ACA AAC TGG 3
• P. falciparum 5 ACA ACC TGG 3
• P. vivax S117T/N
• P. falciparum S108T/N

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The numbers
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Interpretation and Results

• The positive controls, HB3 and K1, verified that
  the BioPlex results were correct, even if the
  numbers are lower than the average output values
• HB3 51A, 59T, 108A, 164A
• K1 51A, 59C, 108A, 164A
• All the field samples had higher levels of
  resistance to sulfadoxine-pyrimethamine than
  wild-type strains
• Since the blood forms of malaria parasites are
  haploid, it can be presumed that the samples with
  more than one positive result from the BioPlex
  machine have more than one parasite strain
  present
• Technique can be used to genotype samples quickly
  and efficiently ? monitor drug resistance in
  different geographical locales

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PNG

• According to Casey et al.
• Prior to his 2004 study, chloroquine had been the
  standard treatment for uncomplicated malaria, and
  Fansidar in combination with quinine was the
  second-line treatment
• During the course of the study, the PNG
  Department of Health revised the standard
  treatment recommendations to chloroquine SP and
  SP artemisinin derivatives
• No polymorphisms were observed at codons 16, 51,
  or 164 of dhfr at either of the two study sites
  (Madang and Maprik)
• There were higher rates of mutations at residues
  59 and 108 in Madang, where people have a higher
  exposure rate to SP
• 108 mutations also strongly correlated with prior
  exposure to primaquine
• Once SP becomes ineffective, all that will be
  left are the artemisinin derivatives

22
Roll Back of Antifolate Resistance

• Studies show that by implementing
  insecticide-treated nets, there is an increased
  frequency of P. falciparum strains carrying the
  wild-type dhfr allele
• ITNs reduce transmission, therby reducing the
  number of drug-exposed parasites

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Future Concerns

• Areas with no drug-resistant malaria
• Efforts are needed to extend chloroquine efficacy
  for as long as possible
• Areas with no multidrug resistance
• Constant surveillance needed for the early
  detection of development of multidrug resistance
• Emerging multidrug-resistant areas
• Prevention and rapid detection of new foci
  development
• New regimens and alternative drugs needed
• Rational drug use must be reinforced to limit
  drug pressure
• Established multidrug-resistant areas
• Efforts needed to limit geographical spread
• New combinations may preserve efficacy of current
  drugs
• Development of new drug candidates needed

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