Current status of Antimalarial Drug Resistance

Current Status of Antimalarial Drug Resistance in Kenya
MAJ Edwin Kamau
Malaria Drug Resistance Program
US Army Medical Research Unit Kenya
MAJ Edwin Kamau (MDR Director)
KEMRI-Walter Reed Project
Disclaimer: The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official or as reflecting the views of the US
Army Medical Research Unit-Kenya or Department of the Army or the Department of Defense.
Funding: This work was supported by the Global Emerging Infections Surveillance and Response System (GEIS), a division at the Armed Forces Health Surveillance Center
(AFHSC).
Malaria Drug Resistance Program
OUTLINE
• Introduction of our organization and how we function
• Emergence and spread of drug resistance
• State of quinine resistance in western Kenya
• State of sulphadoxine-pyrimethamine resistance in Kenya
• Artemisinin resistance
• State of artemisinin resistance in western Kenya
• Conclusion
• There are 24 content slides
Malaria Drug Resistance Program
• US Army Medical Research Unit-Kenya
• USAMRU-K Department of Emerging Infectious Diseases
Our Mission
To conduct surveillance and comprehensive studies of drug-resistanceassociated mutations, genetic lineages and population structure. To
support our partners and collaborators in molecular analysis. To test new
antimalarials compounds.
Vision
To become a world class laboratory in Malaria Drug Resistance studies.
MDR Surveillance Sites
Active sites
Isiolo District Hospital
Malindi District Hospital
Kericho District Hospital
Kisumu East District Hospital
Kombewa District Hospital
Kisii District Hospital
Marigat District Hospital
Soon to be activated Sites (Wish list)
Marsabit District Hospital
Lodwar District Hospital
Mombasa District Hospital
Gilgil district hospital
Inactive Sites (Located on Kenya’s Borders )
Dadaub Refugee Camp (Kenya-Somalia)
Busia Sub-District Hospital (Kenya-Uganda)
Moyale District Hospital (Kenya- Ethiopia)
Kakuma Refugee Camp (Sudan Kenya)
4
Sample Flow and Processing
Field Isolates/Efficacy study
Immediate ex vivo +
in vitro testing
Genetic analysis and
Genotyping
Per-drug IC50s
SNP / copy
Number/sequencing
Correlate in vitro +
molecular data
5
Emergence of resistance
• Predicting the emergence and spread of
resistance to current antimalarial drugs is
important for the planning and execution
of malaria control and intervention
strategies
De novo
appearance of
resistance
• Various genetic mechanisms of resistance
some are not very clearly understood
• Emergence of resistance begins with
random mutations occurring in the
presence of drug treatment, followed by
transmission and stochastic persistence
until the mutation(s) becomes established
in the population
• Could it be natural evolution of the
parasite?
Selection of de
novo resistance
The spread of
resistance
Appearance and selection of de novo resistance
• Selection can occur by exposing a large
number of parasites to sub-therapeutic
antimalarial drug concentration
• Factors determining probability of
selection
Frequency of the resistance mechanisms
Level of host immunity
Fitness cost
Number of parasites exposed to the drug
Concentration of the drug exposure
Pharmacological properties of the drugs
Degree of resistance
Role of MOI?
Human behavior ?
Figure Showing drug response by diverse parasite phenotypes
Spread of resistance
• The recrudescence and subsequent
transmission of an infection that
generated resistant parasites de novo
are essential for resistance to be
propagated
•Transmission of a de novo resistant
mutant out of the primary host is the
largest hurdle that resistant parasites
face
The parasite first must survive the
immune systems
The resistant mutation must not be
lost during meiosis
Must be viable for transmission
Spread of resistance…conti..
• Low and unstable transmission favors the faster emergence of
resistance
• Drug resistance spreads through the high transmission population
much faster than in a low-transmission setting
Implications for the elimination agenda?
• The gametocytes that transmit resistance arise from the subsequent
recrudescent infection
The resistant parasites multiply and produce the gametocytes in the recrudescent
infection
• Spread between populations is mediated by the movement of
individuals and vectors
The rate that drug resistance will spread between populations is a function of the
frequency that resistance is introduced into the new population combined with the
probability of the resistant parasite becoming established
State of Quinine Resistance in Western Kenya
• QN is 2nd-line treatment for uncomplicated malaria, an alternative 1stline treatment for severe malaria, and for treatment of malaria in the first
trimester of pregnancy
• QN has been shown to have frequent clinical failures. Mechanisms
resistance not fully elucidated
• Resistance is linked to polymorphisms in multiple genes; multidrug
resistance 1 (Pfmdr1), the chloroquine resistance transporter (Pfcrt), and
the sodium/hydrogen exchanger gene (Pfnhe1)
• We recently investigated the association between in vitro QN
susceptibility and genetic polymorphisms in Pfmdr1 codons 86 and 184,
Pfcrt codon 76, and Pfnhe1 ms4760 in 88 field isolates from western
Kenya (Cheruiyot et al. 2014)
State of Quinine Resistance in Western
Kenya…conti..
• Data revealed there was significant associations between
polymorphism in Pfmdr1-86Y, Pfmdr1-184F, or Pfcrt-76T and QN
susceptibility
• Eighty-two percent of parasites resistant to QN carried mutant alleles
at these codons (Pfmdr1-86Y, Pfmdr1-184F, and Pfcrt-76T), whereas
74% of parasites susceptible to QN carried the wild-type allele (Pfmdr1N86, Pfmdr1-Y184, and Pfcrt-K76, respectively)
• In addition, QN IC50 values for parasites with Pfnhe1 ms4760
3 DNNND repeats were significantly higher than for those with 1 or 2
repeats
• For all three genotypes (Pfmdr1-86Y, Pfmdr1-184F, and Pfcrt-76T), the
cumulative effect was highest in the background of 3 DNNND repeats,
reaching statistical significance in each one of the genotypes
State of Quinine Resistance in Western
Kenya…conti..
• 18.2% (16/88) of the isolates exceeded the 500 nM cuttoff and 11.4%
(10/88) exceeded the 800 nM cutoff, and were therefore considered
resistance
•These are relatively high number of QN resistant compared to other
locations (studies)
• In studies conducted in Senegal and Kenya (Kilifi), only 1% and 7%
(respectively) of the isolates tested against QN had IC50s of 500 nM
• In the Kilifi study, none of the isolates had IC50 of 800 nM
• In field isolates from the Republic of the Congo, 25.7% exceeded the 500
nM cutoff, whereas only 5.4% exceeded the 800 nM cutoff
State of Sulphadoxine-pyrimethamine Resistance in
Kenya
• Sulphadoxine-pyrimethamine (SP), an antifolate, was replaced by
artemether-lumefantrine as the first-line malaria drug treatment in Kenya
in 2006 due of the wide spread of resistance
• However, SP still remains the recommended drug for intermittent
preventive treatment in pregnant women and infants (IPTP/I) owing to its
safety profile
• We recently conducted a study to assess the prevalence of mutations in
dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps)
genes associated with SP resistance in samples collected in Kenya
between 2008 and 2012 (Juma et al. 2014)
• Field isolates collected from Kisumu, Kisii, Kericho and Malindi district
hospitals were assessed for genetic polymorphism at various loci within
Pfdhfr and Pfdhps genes by sequencing
State of Sulphadoxine-pyrimethamine Resistance in
Kenya…conti..
• Pfdhfr is target for pyrimethamine and Pfdhps is target for sulphadoxine
• Pfdhfr S108N mutation has been linked to pyrimethamine resistance.
Additional point mutations A16V, N51I and C59R lead to increased
resistance
• High grade pyrimethamine resistance is linked to the occurrence of the
I164L mutation
• Pfdhps A437G mutation is mainly associated with sulphadoxine
resistance with increased resistance conferred in the presence of
additional point mutations S436A/F/H, A581G, K540E and A613S/T
• Primarily, the Pfdhfr/Pfdhps N51I, C59R, S108N/A437G, K540E
quintuple mutation has strongly been associated with clinical SP treatment
failure
State of Sulphadoxine-pyrimethamine Resistance in
Kenya…conti..
• Pfdhfr mutations, codons N51I, C59R, S108N showed highest prevalence in all
the field sites at 95.5%, 84.1% and 98.6% respectively
• Pfdhfr S108N prevalence was highest in Kisii at 100%
• A temporal trend analysis showed steady prevalence of mutations over time
except for codon Pfdhps 581 which showed an increase in mixed genotypes
• Triple Pfdhfr N51I/C59R/S108N and double Pfdhps A437G/ K540E had high
prevalence rates of 86.6% and 87.9% respectively
• The Pfdhfr/Pfdhps quintuple, N51I/C59R/S108N/A437G/K540E mutant which
has been shown to be the most clinically relevant marker for SP resistance was
observed in 75.7% of the samples
• When further broken down per site, Kericho and Kisumu had the highest
prevalence at 89.6% and 80.6%, respectively whereas Malindi had the lowest
prevalence at 54.3%
State of Sulphadoxine-pyrimethamine Resistance in
Kenya…conti..
• SP resistance is still persistently high in western Kenya
• This is likely due to fixation of key mutations in the Pfdhfr and Pfdhps genes
as well as drug pressure from other antifolate drugs being used for the
treatment of malaria and other infections
• In addition, there is emergence and increasing prevalence of new
mutations in Kenyan parasite population (Pfdhfr I164L and Pfdhps K540E)
• Since SP is used for IPTP/I, molecular surveillance and in vitro susceptibility
assays must be sustained to provide information on the emergence and
spread of SP resistance
• Sample isolates collected from Malindi have significantly different
prevalence of drug resistance alleles in Pfdhfr and Pfdhps genes compared
to sample isolates collected from western Kenya
Artemisinin Resistance
• WHO has recommended artemisinin-based combination therapies (ACTs) as firstline treatment for uncomplicated P. falciparum malaria since 2001
• Most malaria-endemic countries have shifted their national treatment policies to
ACTs. Kenya shifted to artemether–lumefantrine (AL) in 2006
• AL remains highly effective in most parts of the world, with the exception of
Southeast Asia (SEA)
• WHO recommends more efficacy studies in Africa
• The efficacy of artesunate–mefloquine is lowest in areas where mefloquine
resistance is prevalent such as the Greater Mekong sub-region
• The failure rates of artesunate–sulfadoxine–pyrimethamine are high in regions
where resistance to sulfadoxine–pyrimethamine is high. This ACT remains effective in
countries in which the combination is used as first-line treatment
• Studies indicate dihydroartemisinin–piperaquine is still highly effective in Greater
Mekong sub-region and in Africa
Artemisinin Resistance
• Three methods are used to monitor antimalarial drug efficacy and drug resistance:
1. Therapeutic efficacy studies
2. In vitro tests
3. Use of molecular markers
• Therapeutic efficacy studies allow measurement of the clinical and parasitological
efficacy of medicines and the detection of subtle changes in treatment outcome. They
are considered the gold standard for determining antimalarial drug efficacy
• In vitro/ex vivo assays and molecular markers of drug resistance also play a crucial
role in tracking resistance with an advantage of being robust and sustainable
• In vitro studies track the changes in parasite phenotype whereas the molecular
markers track the change in parasite genotype
• MDR is currently using all the three methods to monitor developments of resistance
Artemisinin Resistance
• Although in vivo efficacy studies are widely used for tracking artemisinin resistance in
SEA, in Africa and other malaria endemic regions, there is a need for concerted efforts to
develop in vitro/ex vivo assays and to identify genetic markers of artemisinin resistance
• Witkowski et al. (2013) described novel phenotypic assay for detection of artemisininresistant P. falciparum parasites, the ring-stage survival assay (RSA0-3 h)
• Evaluation of the P. falciparum genome for regions of recent strong evolutionary
selection and genome-wide association studies revealed regions on chromosomes 10 and
13 that are potential loci involved in artemisinin resistance (Cheeseman et al. 2012,
Takala-Harrison et al. 2012)
• Ariey et al. (2014) showed that RSA0-3 h survival rates and slow parasite clearance
were associated with SNPs in the PF3D7_1343700 kelch propeller domain on
chromosome 13 (K13-propeller)
• K13-propeller mutations were more prevalent in provinces with documented artemisinin
resistance
• Subpopulations of parasites can be categorized as sensitive or resistant based on their
genomic profile (Phyo et al. 2012; Miotto et al. 2013)
State of Artemisinin Resistance in Western
Kenya…conti..
• AL has been associated with selection of single nucleotide polymorphisms (SNPs)
in P. falciparum multidrug resistance 1 (Pfmdr1) and P. falciparum chloroquine
resistance transporter (Pfcrt) genes among re-infections, as compared with baseline
parasite characteristics
• The main SNPs are Pfmdr1 N86, 184F and D1246 (NFD) and Pfcrt K76
• These studies have mostly been conducted in Africa, with a recent study conducted
in Mbita, western Kenya where the selection was seen for both AL and
dihydroartemisinin piperaquine (DP)
• In Africa, NFD can be considered the genetic signature of slow clearing
sub‐microscopic infections in ACT‐treated patients
State of Artemisinin Resistance in Western
Kenya…conti..
• We are currently investigating the prevalence of Pfmdr1 N86, 184F and D1246
(NFD) and Pfcrt K76 in field isolates collected before introduction of ACTs (pre-ACTs)
and after (post-ACTs)
• Pre-ACTs samples collected from 1995-2003 and post-ACTs 2008-2014
• Preliminary data indicate as follows comparing pre and post-ACTs isolates
1. N86 increased from 32% to 100% (p = 0.001)
2. 184F increased from 35% to 65% (p = 0.0016)
3. K76 increased from 6% to 46% (p = 0.001)
• We also have data on in vitro susceptibility of these isolates for DHA, artemether,
lumefantrine, mefloquine and amodiaquine
• We are still working on data analysis, manuscript should be ready shortly
State of Artemisinin Resistance in Western
Kenya…conti..
• We are also conducting ACTs efficacy trial in Seme Sub-County of Kisumu County
based upon the classic WHO antimalarial drug surveillance protocol, but modified to
measure all the key determinants of parasite clearance. Detailed parasite sampling is
conducted during the initial period of treatment
• Drug pharmacokinetics and pharmacodynamics are key determinants of parasite
clearance, and hence treatment outcome. Other important factors include parasite
genetic characteristics, parasite load, innate host resistance to malaria and acquired
immunity to malaria
• Frequent parasite sampling is being done, together with sparse pharmacokinetic
sampling
• Additionally in vitro sensitivity testing and genetic testing of the parasite samples is
being carried out
• Immunology testing will be done to complement the findings of the in vivo and in
vitro testing
State of Artemisinin Resistance in Western
Kenya…conti..
• We have two efficacy studies underway. We are in the process of concluding the
first study where 118 subjects were successfully enrolled
• The study treatments were AL and artesunate-mefloquine (ASMQ)
• In order to accurately evaluate the artemisinin derivative without the confounding
influence of the partner drug, ASMQ was sequentially administered
• Data from ASMQ arm will be compared to data being generated from similar studies
that are underway in Thailand and Peru
• In vitro and genetic data analysis is underway
• Preliminary data analysis show that good ACTs therapeutic response is still
maintained in western Kenya
State of Artemisinin Resistance in Western
Kenya…conti..
• We are set to start the second study within the next few days in the same location
as the first study
• Subjects will be randomized to receive either AL or DP
• The study targets to enroll 59 subjects per arm
Conclusion
• We are focused on using all the three methods of tracking drug resistance where
possible
• We plan to conduct more efficacy studies
• We are continuing to expand and use new strategies in our surveillance effort
• We have a new focus of doing more detailed genetic profiling and population
structure studies. We have introduced new technologies and expanded our
knowledge
• We are expanding our collaborative effort, we are now part of African Plasmodium
Diversity Network (PDNA)
Acknowledgements
Malaria Drug Resistance Team
The Molecular Group
The In vitro Group
The Clinical efficacy Group
Field Sites Staff
GEIS Team
Duke Omariba
Hospital Site Administrators
USAMRU-K Support Staff
Division of Malaria Control

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