Antimalarial treatment and malaria transmission:insights from the field
Abdoulaye DJIMDE PharmD, PhD
Malaria Research and Training Center
University of Bamako, Mali
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Atelier PaludismeInstitut Pasteur Antananarivo
14 – 20 Mars 2011
2
• Drug Resistance
• Spread of drug resistance
Molecular Biology
Genetics
PK
Pharmacogenomics
Immunology
Policy
Host-Parasite-Vector
Transmission
Outline
1. Sulfadoxine-Pyrimethamine (SP) and malaria transmission in the field
2. Impact of SP on P. falciparumgametocytes infectivity in vitro
3. Artemisinin-based combinations and malaria transmission in sub-Saharan Africa
Conclusion3
P. falciparum life cycle
Current malaria control tools
• Artemisinin-based combination therapies (ACTs)
• Sulfadoxine-pyrimethamine recommended for IPT in pregnant women– contemplated for IPTi and IPTc
• Quinine for severe malaria
• Insecticide treated nets & indoor residual spray
Sulfadoxine-Pyrimethamine treatment and malaria transmission in a setting of
high Sulfadoxine-Pyrimethamine efficacy of Mali
Background
• Chloroquine efficacy decreasing
• Need alternative second line drug
• Prospective in vivo tests of CQ, amodiaquine and SP
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Study site
• Kolle: rural village; 2,500 people
• 55 Km South of Bamako
• P. falciparum malaria endemic and seasonal
• Parasitemia:
– 40-50% dry season (October-April)
– 70-85% rainy season (May-September). Kolle
Study design
• Open randomized drug efficacy trial
• Children 6 months – 5 years
• Three arms: Chloroquine, Amodiaquine, SP
• 28 days of follow up
• In vivo, in vitro , molecular and pharmacokinetic studies
MIM Antimalarial Drug Resistance Network
In vivo SP resistance in Mali
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Evolution of gametocyte carriage after SP treatment
Beavogui et al., IJP 2010
Molecular markers of SP resistance
Adapted from P. Wang et al. :Molecular and Biochemical Parasitology 89 (1997) 161–177
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Mutations in pre-treatment asexual vs. post-treatment gametocytes
0102030405060708090
100
%
DHFR108
DHFR59DHFR51DHPS43
7FRTrip
leQua
drup
le
AsexualGameto
*
**
* * p<0.001*
*
Beavogui et al., IJP 2010
Impact of large scale use of SP on spread of drug resistance and
malaria transmission?
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Methods
• Drug efficacy study• Screening for gametocyte carriers • Detect molecular markers of drug resistance• Include gametocyte carriers aged 6 – 18 y.• Direct feed starved F1 generation An. gambiae• Maintain mosquitoes in lab for 8 days• Presence and number of oocysts measured by
dissection• Compare the infectivity of pre-treatment vs. post-SP
gametocytes to Anopheles gambiae• Protocol approved by IRBs in Bamako & Maryland
16
17
Direct feeding
18
19
Infectivity of Post-treatment Gametocytes
* P < 0.001
Beavogui et al., IJP 2010
Summary 1
• SP increase rate of gametocyte carriage
• Post-SP gametocytes carry SP- resistance mutations
• Post-SP gametocytes were lesstransmissible to Anopheles gambiae
Mechanism of decreased infectivity of post-sulfadoxine-pyrimethamine P. falciparum
gametocytes to anophelinemosquitoes
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Infectivity of Sulfadoxine-Pyrimethamine treated P. FalciparumGametocytes to Anopheline Mosquitoes
Candle Jar Shaker Tipper
• Serum supplemented RPMI complete culture media
• Gas (O2, CO2 and Nitrogen) humidity (70 - 80%)
Gametocyte production
Gametocytes followup
Stage II
Day 8
Stage V
Day 14
Exflagellation test
Day 14
Standard Membrane feeding Assay
Feeding of 30 mosquitoes
With each sample
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Sulfadoxine and Pyrimethamine Plasma concentrations in Mali
S
P
Drugs Mean conc. Day 3 Day 7 Day 14
Sulfa -------
Pyr
ug/ml------------ng/ml
61-----------
158
34------------
67
14---------
16
Drug concentrations used
Experimental design
28A = Gametocytogenesis, B = Gametocyte maturation, C = Mature gametocyte infectivity Kone A, IJP, 2010
Results
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A. Induction of gametocytogenesis
30NF 135 Stage II NF135 Stage V
B. NF 135 Gametocyte development
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Controls Pyrimethamine- treated Sulfadoxine- treated
Kone A, IJP, 2010
C. Effect of S, P and SP on gameto infectivity
32Kone A, IJP, 2010
Effect of S, P and SP on oocyst density
33Kone A, IJP, 2010
Effect of Day3 levels of S or SP on mosquito survival
34Kone A, IJP, 2010
Summary 2
• Sulfadoxine and pyrimethamine have complex effects on the biology of gametocytogenesis.
• Induce differentiation into sexual forms
• Treatment of young gametocytes impaired their further development into mature stage V gametocytes.
• Drug + mature gametocytes => decreased infectivity
• SP also kills vector A. stephensi35
Artemisinine-based combination therapies and Malaria transmission
in the field
ACTs and malaria transmission
• “A single intramuscular injection of 5 mg/kg artemisinin (to gametocytemic rhesus monkeys) resulted in complete loss of mosquito infectivity within 24 h of drug administration” Dutta GP, et al. 1989
• “artemisinin derivatives reduced gametocyte carriage 18.5 fold”and “were found to reduce the transmission potential of falciparum malaria”Price RN et al.,1996
• “Artemesinin-based combination therapies (ACT) for falciparum malaria reduce gametocyte carriage, and therefore reduce transmission”.
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ACTs and malaria transmission (2)
• NASBA study in Kenya “data suggest that the potential of malaria transmission remains high even after treatment with artemisinin combination therapy”Schneider P. et al, IJP, 2006
• “An efficacious antimalarial regimen with no specific gametocytocidal properties but a long prophylactic time was estimated to be more effective at reducing transmission than a short-acting ACT in the highest-transmission setting”. Okell LC, PLoS Med, 2008
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Objective
Measure the impact of AS/AQ, AS/SP and AR-L on malaria transmission.
Day 28 Efficacynon-Corrected vs. PCR Corrected
** P < 0.05
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Evolution of gametocyte carriage by treatment arm on follow up days
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How infectious are the post-ACT gametocytes?
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Methods• Drug efficacy study• Screening for gametocyte carriers • Detect molecular markers of drug resistance• Include gametocyte carriers aged 6 – 18 y.• Direct feed starved F1 generation An. gambiae• Maintain mosquitoes in lab for 8 days• Presence and number of oocysts measured by dissection
• Compare the infectivity of pre-treatment vs. post-SP gametocytes to Anopheles gambiae
• Protocol approved by Ethics Committee of FMPOS
Study site
• Bougoula-Hameau: peri-urban village; 5000 people
~400 Km South of Bamako
• P. falciparum malaria hyper endemic
• No Insectaries around!!
Bougoula-Hameau, Sikasso, Mali
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46
47
48
49
Direct feeding
50
51
Comparing Ctrl vs. all post-ttt Oocyst positive
05
1015202530354045
Ctrl
ASSP
ASAQ
ARL
Treatment arms
Oocyst +
***
***
NS
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Summary 3
• ACTs decreased gametocyte carriage
HOWEVER
• Would all ACTs reduce malaria transmission in high transmission settings?
Discussion
• “Overall, infectivity was about three-times higher for direct feeding than for membrane feeding (p < 0.001)”Diallo M., et al Malaria Journal 2008
• MFA “Blood cells were separated from plasma by centrifugation and plasma was replaced by a similar volume of normal human plasma known to sustain malaria transmission. This step was performed to avoid the possible interference (blocking or enhancing) antibodies to gametocytes in the donor’s plasma”.
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Conclusions
• Malaria eradication/elimination will require new, safe and truely gametocytocidal drug
• Need more sensitive gametocyte assays that could indicate gametocyte viability in addition to prevalence and density.
• Field oriented studies need to be as close as possible to natural conditions. Too much cleaning of experimental design may yield nice results but with little relevance to real life. 54
Acknowledgements• MRTC
• Study sites & participants
• Pr. Doumbo O &
MRTC staff
• Nijmegen
• Adrian Luty
• Robert Sauerwein
• Support
• Government of Mali
• MIM/TDR
• NIAID/NIH
• FIC/NIH
• Ministère Français de la Recherche (Pal +)
• Sanofi-Aventis
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