Horn of Africa Network, Cairo, Egypt

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Overview of various methods used in monitoring
(in vivo, in vitro, molecular markers)

Horn of Africa Network, Cairo, Egypt 22-25 March
2004
Dr P. Ringwald Roll Back Malaria Department
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What is antimalarial drug resistance?

• Ability of a parasite strain 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
  tolerance of the subject (WHO, 1973).
• The drug must gain access to the parasite or the
  infected red blood cell for the duration of the
  time necessary for its normal action (WHO, 1986).
• Drug resistance ? treatment failure
• (host and/or parasite factors)

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Chemical families Drugs

• 4-Aminoquinolines
• Arylaminoalcohols
• Sulfonamides/sulfones
• Biguanides
• Diamino-pyrimidine
• 8-Aminoquinolines
• Sesquiterpene lactones
• Naphthoquinones
• Antibiotics

• Chloroquine, amodiaquine
• Quinine, quinidine, mefloquine, halofantrine,
  lumefantrine
• Sulfadoxine, sulfalene, dapsone
• Proguanil, chlorproguanil
• Pyrimethamine
• Primaquine
• Artemisinin, dihydroartemisinin, artesunate,
  artemether, arteether
• Atovaquone
• Tetracycline, doxycycline, clindamycin,
  fosmidomycin

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Antimalarial Year of First case
drugs introduction of resistance

• Quinine
• Chloroquine
• Proguanil
• Sulfadoxine-pyrimethamine
• Mefloquine
• Atovaquone

• 1632 1910
• 1945 1957
• 1948 1949
• 1967 1967
• 1977 1982
• 1996 1996

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Malaria distribution and reported case of
resistance
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Consequences of antimalarial drug resistance

• Increased morbidity and mortality including
  anaemia, low birth weight...
• Increased gametocyte carriage
• Economic impact
• Greater frequency and severity of epidemics
• Modification of malaria distribution

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Combating antimalarial drug resistance

• Reducing the disease burden and the spread of
  infection
• Improving diagnosis, prescribing, and use of
  antimalarials
• Improving access to appropriate antimalarials
  (ACTs)
• Development of new drugs
• Strengthening health systems in their
  surveillance capabilities
• Enforcing regulations and legislation
• Encouraging research

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Tools for monitoring drug resistance

• Ideal situation 2 tools
• Primary tool
• Continuous early warning system with definable
  critical level/threshold ? triggers secondary
  tool
• Secondary tool
• Periodic in depth assessment of clinically
  relevant information

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Tools for monitoring drug resistance

• THERAPEUTIC EFFICACY TESTS
• Standardised protocols for high and
• low/moderate transmission areas
• inclusion criteria, follow-up 14-28 d
• New classification
• ACPR, LPF, LCF, ETF
• clinical /- parasitological criteria
• Limitation
• (degree of) immunity
• prior/concomitant Tx
• PK/PD

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Tools for monitoring drug resistance

• IN VITRO TESTS
• Lab equipment, training
• Lack of standardisation
• WHO, isotopic, pLDH
• Multiple tests to determine
• cross-resistance
• Provide baseline data and
• trends
• Forecast (drug for the future)
• Precede in vivo resistance
• Threshold of resistance to
• be validated

• THERAPEUTIC EFFICACY TESTS
• Standardised protocols for high and
• low/moderate transmission areas
• inclusion criteria, follow-up 14-28 d
• New classification
• ACPR, LPF, LCF, ETF
• clinical /- parasitological criteria
• Limitation
• (degree of) immunity
• prior/concomitant Tx
• PK/PD

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In vitro isotopic microtest
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Correlation between lumefantrine and mefloquine
or halofantrine
100.0
10.0
1.0
0.1
1
10
100
lumefantrine
IC
50
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Correlation between pyrimethamine and cycloguanil
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In vitro susceptibility of 8 antimalarial drugs
IC50 values (nmol/l)
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Geographical trend
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In vitro Pros

• In vitro tests reflect drug resistance
• in direct testing on P. falciparum parasites
• in vivo tests reflect therapeutic efficacy
• eliminate host factors
• Quantitative results
• compared between sites and time if consistent
  method
• Multiple test on same isolates
• Several drugs can be assessed simultaneously
• Experimental drugs can be tested
• high drug concentrations
• Precede in vivo resistance

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In vitro tests cons

• Lack of correlation with clinical response
• presence of parasites with different drug
  sensitivity phenotype
• re-infection with new populations of parasites
• pharmacokinetic of the drug
• acquired immunity
• self medication or previous drug intake
• Prodrugs and combination
• difficult to test
• Biological advantage of resistant isolates
• introduction of bias

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In vitro tests cons

• Expensive and technically difficult
• Except for Mark III
• Some tests need a large amount of blood
• Technical issues
• Lack of standardization
• Cut-off values or threshold of resistance not
  validated
• Others species

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Tools for monitoring drug resistance

• IN VITRO TESTS
• Lab equipment, training
• Lack of standardisation
• WHO, isotopic, pLDH
• Multiple tests to determine
• cross-resistance
• Provide baseline data and
• trends
• Forecast (drug for the future)
• Precede in vivo resistance
• Threshold of resistance to
• be validated

• THERAPEUTIC EFFICACY TESTS
• Standardised protocols for high and
• low/moderate transmission areas
• inclusion criteria, follow-up 14-28 d
• New classification
• ACPR, LPF, LCF, ETF
• clinical /- parasitological criteria
• Limitation
• (degree of) immunity
• prior/concomitant Tx
• PK/PD

• MOLECULAR MARKERS
• Differentiation between
• recrudescence and re-infection
• Correlation between mutations
• and in vivo/in vitro data
• Lab equipment, training, standardisation
• Multiple tests/drugs
• Provide baseline data and
• trends

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Treatment Failures
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Treatment failure under endemic conditions
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True recrudescence
Dx
D0
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Multiple genetic markers
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True recrudescence or re-infection?
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Plasmodium pyrimidine pathway
CO2 aspartate
dihydroorotate
ubiquinone H2
cytochrome
1/2 O2
DHOD
ubiquinone
orotate
cytochrome H2
H2O
UDP
dUTP
GTP
dUDP
dTMP
DNA
TS
pteridin
dUMP
pABA
DHPS
dihydropteroate
DHF
N5,N10-CH3THF
THF
DHFR
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MECHANISMS OF RESISTANCE Antifolate drugs
dihydroneopterin
sulfonamides, sulfones
dihydropteroate
dihydrofolate
pyrimethamine, biguanides
Compete for active site with DHF
DHFR (TS)
tetrahydrofolate
Met Gly dTTP
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59
51
Drug/substrate
108
Bolivia repeat
NADP
16
164
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P. falciparum dihydrofolate reductase
Ser108Asn
Ala16Val
Ile164Leu
Cys59Arg
Asn51Ile
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pfcrt gene product with 10 transmembrane domains
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PCR/RFLP analysis of chloroquine resistance
marker pftcrt T76
M 1 2 3 4 5
Dd2 3D7 C M
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MECHANISMS OF RESISTANCEOther drugs
Compound class Drugs Gene Codon with
AA change
4-aminoquinolines Chloroquine pfmdr1 86 (1034,
1042 1246) cg2 12
codons repetitive regions
pfcrt 76
Amino-alcohols Mefloquine pfmdr1 copy number?
Halofantrine 86, 1034,
Quinine 1042, 1246
Sesquiterpene lactone Artemisinin pfmdr1
86, 1034, 1042, 1246
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Relation between chloroquine IC50s and K76T
mutation
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Relation between pyrimethamine and cycloguanil
IC50s and mutations
100000.0
10000.0
1000.0
100.0
10.0
1.0
0.1
PYR
CYC
PYR
CYC
PYR
CYC
PYR
CYC
PYR
CYC
Wild-type N108 N108R59
N108R59I51 mixed
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Relation between cycloguanil IC50 and mutations
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Molecular markers

• Pro
• Easy to obtain a large number of samples on
  filter paper
• Cons
• Mutations for a small number of drugs
• CQ, PYR, SDX, atovaquone
• Identification of an epitope close to resistance
  epitope
• Mixed infections

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Molecular markers future

• Validation of markers
• genotype resistance index (GRI) prevalence
  genotype/prevalence clinical/parasitological
  failure
• epidemiological settings
• Identification of markers for other drugs
• Malaria species other than P. falciparum
• Centres of reference