Monitoring of antimalarial resistant

Plasmodium falciparum phenotype

and genotype through complete
life cycle in humanized mice
Irfanul chakim
PhD.Sc student
College of public health sciences
Chulalongkorn university
1
Malaria remains a
major public health
problem worldwide
with an estimated
214 million cases and
438.000 deaths in
2015 (WHO 2015)
2
Plasmodium vivaxPlasmodium Plasmodium ovale
malariae

Plasmodium Plasmodium
falciparum knowlesi

It is primarily caused by
Plasmodium parasite
transferred by female
Anopheles mosquito.
There are five
Plasmodium known to be
a causal agent for
malaria in human host,
namely, Plasmodium
falciparum, Plasmodium Malaria-infected
vivax, Plasmodium person
malariae, Plasmodium
ovale and Plasmodium
knowlesi (
 The complexity of Plasmodium life cycle along with
emergence of drug resistance and lack of understanding to
host immune responses have complicated the eradication
efforts all the time (Miller L.H., Baruch D.I. et al. 2002 ;
Liwang C., Sungano M. et al. 2015)

3
 Background
Malaria
control
strategy

insecticide- effective
impregnated antimalarial drug
bednets and indoor- treatment against
residual spraying of the parasite
insecticides

4
Quinoline
Quinine Resistant
Antimalarial drugs
Chloroquin Resistant
e
Amodiaquin Resistant
e
Mefloquin Resistant
e
Piperaquin Resistant
e
Primaquin ?
e
Lumefanthri ?
ne
Antifolates Sulfadoxine 5
Resistant
Phyremetham
ine

Artemisini Derivatives Resistant

n

Antibiotics Second line treatment

6
Genetic marker
pfcrt Amodiaquine, Quinine,
Lumefanthrine, and Chloroquine

Lumefanthrine, Artemisinin,
pfmdr1
Quinine, Mefloquine,
Halofanthrine and Chloroquine

pfmrp Chloroquine and Quinine

pfnhe Quinine

pfdhfr/pfdh Sulfadoxine and Pyrimethamine

ps

Artemisinin
K13
 Humanized mice
This humanized mice technique
offer a better opportunity to
generate specific strain of
parasite and thus will complete
our gap of knowledge to this
kind of species.
Immunodeficient mice engrafted with human cell, offer a cost-effective and
easily manipulated laboratory model to study human-restricted pathogens
in vivo (Legrand N. and al 2009). To date, such mice engrafted with human
hepatocytes (hHEP) or human RBCs (hRBC) have been shown to sustain
P. falciparum hepatic development and the multiplication of blood stages (
Moore J.M., Kumar N. et al. 1995; Moreno A. and al 2006; Morosan
S. and al 2006; Sacci Jr. J. B. and al 2006; Angulo-Barturen I. and al 2008;
Arnold L. and al 2011). Soulard et al. have shown improvement on previous
protocols to achieve efficient double engraftment of TK-NOG mice by
human primary hepatocytes and red blood cells (Valerie Soulard, Henriette
Bosson-Vanga et al. 2015).

7
 Initial studies of whole genome
sequencing on Plasmodium genus is
particularly important to commence both
practical and latest constructive data for
understanding complex genetic
architecture of Plasmodium genus
8 evolving drug resistance phenotype by
selection pressure of environmental factor
Eventually, these particular latest
technologies of whole genome sequencing
that can produce a large data set of
comparative genome will be suitable for
this study because, compared to genome
association study (GWAS) that adopt the
common disease- common variant
(CDCV) hypothesis, linkage
disequilibrium and population structure of
sample being studied will not be took into
account and therefore no indication of
false positive and genotyping error like
GWAS does.

Genome wide scale

Research problem

To the past century, introduction of several

antimalarial drugs have seen to be a promising
strategy to eradicate malaria. However, in line with
elevated number of publication and the more better
technique and methodology, there are plenty of
reports have made evidences the emergence of
Plasmodium resistant to these drugs. Moreover, the
discovery of gene-specific candidate that would be
useful in understanding Plasmodium reistant isolates
tends to be conflicted by cross resistance and
overlapping mutation. Therefore, with a new
knowledge to generate full life cycle of Plasmodium
malaria in vivo, it is will be possible to generate
strain specific resistant parasite to validate
population based study. Additionally, the presence of
next generation sequencing which can read whole
genome sequence offer a better opportunity to
analyze the full genome of these engineered
Plasmodium falciparum.
9
Research questions and objectives
1. Can Plasmodium
falciparum resistant strain
be established through a
complete life cycle
strategy and continuous
antimalarial drug
pressure?
2. How is the genotype and
phenotype profile of
Plasmodium falciparum
resistant strain which is
produced through
complete life cycle
compared to the sensitive
strain?
3. Is there any cross
resistance among individu
convering specific
10 resistant to a particular
1. To investigate the successful
establishment of Plasmodium
falciparum convering resistant
genotypically and
phenotypically through a
complete life cycle and
continuous antimalarial drug
pressure
2. To investigate the genotype
and phenotype profile of
Plasmodium falciparum
resistant strain which is
produced through complete
life cycle compared to the
sensitive strain
3. To investigate the occurrence
of cross resistance among
individu convering specific
resistant to a particular
Expected benefit/application

The expected result of this study is providing a

resistant-specific parasite strain to be a validating
strain from laboratory or field based study. The
absence of this specific-parasite strain was the
paramount limitation of researchers work when
discovering certain mechanism(s) mediating
resistance to a particular drug regime. Secondly,
genetically inheritable phenotype specific to a
particular strain of parasite convering specific
resistant to a drug regime is a crucial stepping
stone for incriminating genetic basis of a certain
drug regime and validating cross-resistance
phenotypically and genetically.

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Limitation

The paramount limitation of this study is the

homogeneity of sample, since this is laboratory
based isolate which can not mirroring filed
circumtances. Repeated mating or engineered
mating from these homogeneity sample also
lead to severe selective pressure and therefore
increasing linkage disequilibrium value.
Eventually, inferential induction of this study
can not dirrectly be generated on population
based study that hold hardy weinberg
equilibrium equation although limited number
of resesive allele exist or not constantly in an
equilibrium phase.

12
13

In vitro cultivation In vivo cultivation

Drug pressure TK-NOG
Temporarily resistant P.
Infect
falciparum

Plasmodium falciparum An. gambiae

Repeated cycle to F-?

A particular F-?
Molecular monitoring
Generation that
Drug susceptibility
have managed to
testing
generate strain
Micromanipulation for
specific
F-1 generation
plasmodium
resistant to
certain drugs
Whole genome
sequencing
Research methodology

14
Properties of study
15

Group Antimalarial drugs Source of the

drugs
The parasite TK-NOG mice- A particular strain of Chloroquine WHO micro-test kit
Amodiaquine WHO micro-test kit
used is from laboratory strain will mosquitoes in this Mefloquine WHO micro-test kit
university of be obtained from Dr. study would be used is Piperaquine Yick-Vic Chemicals
Tokyo, that Valerie soulard from Anopheles gambiae. and Pharmaceuticals
Quinolines
have been Sorbonne universites, This strain has been Ltd. (Kowloon, Hong

propagated France. These TK-NOG widely reported due to Kong)

Lumefanthrine Sigma-Aldrich Co.
regularly under mice was double its major role of
LLC
bacterial-free engrafted by primary transmitting malaria in Quinine WHO micro-test kit
strict condition human hepatocytes Africa and widely Sulfadoxine/phyrime WHO micro-test kit
Antifolates
in Eijkman and red blood cells, distributed throughout thamine
Arthemeter Adooq Bioscience
institute, therefore have made the afro-tropical belt. Artesunate Sigma-Aldrich Co.
Jakarta. it successfully Therefore, with known LLC.
Artemisinin
generated to mirror ability to transmit Dihydroartemisinin Dafra Pharma NV
human blood plasmodium, this (Turnhout, Belgium)
Artemisinin WHO micro-test kit
circulation system and species would be
enable the potentially used in this
plasmodium study as a vector in a
Procedure The origin of humanized mice
NOD/Shijic- C57BL/6JJic-
Prkdcscid IIrg

NOG TK-NOD/Shi

TK-NOG

16
Procedure The origin of humanized mice

Description of TK-NOG mice

Useful for humanizing liver via engraftment with human hepatocytes and/or humanizing immune
system via engraftment of human immune cells/tissues or hematopoietic stem cells
Inducible ablation of murine hepatocytes by ganciclovir treatment enables stable, long-term
engraftment of human hepatocytes.
The premiere model for human liver toxicity, metabolism, and infectious disease.
Super immunodeficient NOG mouse with transgenic expression of thymidine kinase under
control of liver-restricted albumin promoter
Other uses include safety and immunogenicity assessment

17
Procedure Anophele gambiae rearing procedur
Day 1 :
Mosquito food:
Day 13-15 :
Food B: grounded CAT food (Purina). The 3-5 day old
adult female
Food C: cat food (Purina). Preparation of adult
mosquitoes are fed
Food A: grounded fish food (Aquaricare). emergence from
on blood to lay
pupae
Blood: eggs.
Human/animal blood. RBC is
Day 3 :
resuspended in serum (O+ Day 16 :
human serum) to obtain a 40%
Laying eggs
haematocrit. The blood must be Collection of adult
medium
kept at 37C always for the mosquitoes
preparation
feeding.

Day 4 : Day 17-21:

Collection of eggs Maintenance of

adult mosquitoes
and repletion of
Day 5-12 : cycle will be begun

Maintenance of
18 larvae that have (Suchismita Das,
2007)
hatched from eggs
Procedure Procedure to generate Anopheles gambiae mosquitoes infected
with Plasmodium falciparum

1.Three main steps:

Plasmodium falciparum gametocyte, culture and cultivation
Maintaining the parasitemia <1% (subculturing).
6-8 days post-subculture stage I and II gametocyte
14 day post-subculture mature, stage V, gametocyte
Day 17 post-subculture proportion of stage V gametocyte in PRBC may be up to
4%, and gametocytemia of all stages of development (I-V) may be up to 12%.
Enrichment of gametocyte is not necessary. It necessitates to mix cultures of
different ages at the time of blood feed.
2. Membrane feeding gametocyte to mosquito
Blood feed the mosquitoes (3-5 days postemergence) with gemtocyte containing
19
membrane feeder which is previously well prepared.
3. Production of Plasmodium falciparum oocysts and sporozoites
The objective of this study is to perform inheritable genotype through continuous
breeding of the parasite complete cycle, thus successive transmission to the
reservoir (The mice in this case) is merely the maint point of this study. Therefore,
there are two types of transmission that can be carried out, which will be explained
in this section: 1. Through direct transmission from infected mosquitoes to the TK-
NOG mice.; 2. Through indirect transmission. This is simply done by transmitting
liberated sporozoites from mosquitos salivary gland into the mice body through
intravenous injection.
All these transmission strategy will be previously confirmed for the presence of
Procedure Procedure of antimalarial susceptibility testing

A-H =
indication
of different
drug that
have to be
dosed 1-12 =
Blood medium
concentration of
micture
drugs, which has
tendency to be
higher as indicated
by increased
number

20 Drug(s
)
 Procedure of molecular marker
Procedure

Gene of interest Amplification technique

1. pfcrt Nested PCR
RT-PCR

2. pfmdr1 RT-PCR
Common PCR

3. pfmrp1 Nested PCR

4. pfmrp2 Nested PCR
5. pfnhe Microsatellite
6. pfdhps Nested PCR
7. pfdhfr Nested PCR
8. K13 Common PCR
9. plasmepsin II RT-PCR

21
Procedure Procedure of molecular marker (Cont.)

22
Procedure Whole genome sequencing

2
3

23
Data analysis Antimalarial susceptibility assay
Counting procedure for non Test drug Satisfactory response Indication of
SYP-PYR resistance
As basis of counting: Number Complete schizont Schizont formation at
of schizonts with three or inhibition at
more nuclei out of a total Chloroqiune 4 pmol or less 8 pmol or more
of 200 asexual parasites Mefloquine 16 pmol or less 32 pmol or more
Quinine 128 pmol or less 256 pmol or more
(i.e. schizonts and Amodiaquine 2 pmol or less 4 pmol or more
trophozoites). Piperaquine - 140 nM or more
Lumefanthrine - 150 nM or more
Counting procedure for SYP- Arthemether - 209.4 nM or more
Artesunate - 10.5 nM or more
PYR
Dihydroartemisinin - 10.5 nM or more
As basis of counting: the *Interpretation of result
schizont growth threshold
has been rearranged to
one of eight or more
normal nuclei. This is due
to unrelevant calculation
based on previous
definition, since the drug
may affect nuclear
24 division.
 Test A
Schizont counts (per 200 parasites) in well

B C D E F G H
Data analysis
number Control 1 pmol 2 pmol 4 pmol 8 pmol 16 pmol 32 pmol 64 pmol
Antimalarial susceptibility assay (Cont.)
As percentages of Control:
Parameter Well
1 100.0 86.3 42.6 22.6 5.8 1.1 0.0 0.0
A B C D E F G H
2 100.0 57.1 28.6 0.0 0.0 0.0 0.0 0.0 Schizonts 55 56 47 8 0 0 0 0
3 100.0 100.6 89.9 77.5 62.4 37.6 19.1 0.0 with 8 or

4 100.0 98.0 96.9 94.9 56.1 33.2 11.7 0.0 more normal
nuclei
5 100.0 08.2 42.9 22.5 1.0 0.0 0.0 0.0
Other 145 144 153 192 200 200 200 20
6 100.0 94.1 88.2 61.8 17.6 2.9 0.0 0.0 schizonts and
0
7 100.0 95.5 55.5 32.5 19.0 5.5 1.5 0.0 rings
8 100.0 44.8 10.3 0.0 0.0 0.0 0.0 0.0
% schizont 100 85 15 0 0 0 0
maturation (%
9 100.0 50.0 23.4 12.5 1.6 0.0 0.0 0.0
of control)
10 100.0 96.4 85.5 47.3 21.6 0.0 0.0 0.0 % inhibition 0 15 85 100 100 100 10
TOTAL 1000.0 831.0 563.8 371.6 185.3 80.3 32.3 0.0 of schizont
0
Total/n =% 100.0 83.10 56.38 37.16 18.53 8.03 3.23 0.00 maturation
maturation
(mean)

% inhibition (0) 16.90 43.62 62.84 81.47 91.97 96.77 100.0

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Data analysis Sequence analysis
1. PfCRT 4. PfMRP2
Codon 72 73 74 75 76 198 220 271 326 356 371
positio
n Nucleotide Nucletide Amino acid Amino acid

3D7 C V M N K E A Q N I R position change position change

561 TA 187 Synonimous
Vast C V I E T E S E S T I
595 CG 199 LV
major
ity 884 CG 295 TR
930 GT 310 LF
18% C V I E T K S E S T I
981 CT 327 Synonimous
4% C V I E A E S E S T I
1777 AG 593 ND
2. PfMDR 1864 AG 622 ND
Codon position 2141 AT 714 KI

Isolate 86 184 1034 1042 1246 2397 CT 799 Synonimous

3414 TC 1138 Synonimous
3D7 N Y S N D
4579 TA 1527 ST
Mutant Y F C D Y
4591 CA 1531 LI
5112 TC 1704 Synonimous
3. PfMRP1
5251 AT 1751 NY
Codon position 5258 TC 1753 FS
Isolate 191 437 876 1390 1466 5323 AG 1775 IV
3D7 H S I F K 5344 AT 1782 ML

Mutant Y A V I R 5385 AT 1795 Synonimous

26
Data analysis Sequence analysis (Cont.)
5. PfNHE
Isolate No. of DNNND No. of Profile 7. K13 propeller
repeats DDNHNDNHNN domain
K13 mutation Classification
repeats E25Q Not associated
3D7 1 2 X*
P441L Candidate
Mutant - - -
F446I Candidate
G449A Candidate
6. Pfdhps and N458Y Validated
Pfdhfr Y493H Validated
Isolat Dhfr Dhps R539T Validated
e 16 51 59 108 164 436 437 540 581 613
I543T Validated

V1 A I R N I F G K A T P553L Candidate
W2 A I R N I F G K A S R561H Validated
V568G Candidate
P574L Candidate
A578S Not associated
C580Y Validated
A674V Candidate

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Data analysis Copy number analysis (Cont.)
A. Absolute quantification
Construction of standard curve is based on the logarithm of the initial copy number of the
standards plotted along the x-axis and their respective Ct values plotted along the y-axis.
1. PfCRT The linear regression line equation is as follows: [Y= mX + b, or Ct = m (log quantity) + b].
According to the linear regression equation, the following equation can be used to
determine the quantity of an unknown sample:

2. PfMDR Nn = 10 ( ), where n = Ct
Quantity = 10 ( )

3. B. Relative quantification
After initial quantification of both target and reference gene, then we can
PlasmepsinII calculate the 2-2Ct (Livak) Method: Note: The efficiency of both gene need to be
near 100% or 5% of each other
Fist, The target gene Ct value can be normalized to that of the reference (ref)
gene, for both the test sample and the calibrator sample:

Ct (test) = Ct (target, test) Ct (ref, test)

Ct (calibrator) = Ct (target, calibrator) - Ct (ref calibrator)
Second, the Ct of the test sample can be normalized to the Ct of the
calibrator:
Ct = Ct (test) Ct (calibrator)
Finally, calculate the expression ratio:
2- Ct = normalized expression ration

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Data analysis Statistical analysis
1. IC50
calculation
IC50 can be calculated by a serial dilution of drug concentration against parasite
viability. The result of inhibition activity test of P. falciparum is then analyzed by
using linier curve between sample concentration with parasite viability in order to
obtain IC50 value.
IC50 value calculation based on standard curve is as follows:
Y = m ln(x) + b
m ln(x) = b-Y
ln (x) =
x (IC50) =

2. Correlation between reduced susceptibility and gene

copy number
Normality of the data is assessed by using Kolmogorov smirnov (>50 samples)
or saphiro wilk (< 50 samples). Correlation of the reduced susceptibility and
gene copy number is measured by using one way ANOVA, in case all the data
is normal. If normality of the data is violated, therefore correlation will be
measured by using kruskal wallis test. All the test would be done with 5%
assumption of confident interval.
29
Data analysis NGS data analysis

1. Examining the quality of reads

2. Genome assembly 3. Genome annotation

30
THANK
YOU
Supplementary material
A1= contains Microinjecto
MCM+diverse r
parasitized red blood Needle +
cells MCM
And the rest just contain
MCM while separation of
individual parasite is
carried out.
Supplementary material

Prkdcscid mice can accept allogenic and xenogenic grafts that making them an
ideal model for cell transfer experiment because of their characterization of
an absence of functional T and B cells, lymphopenia,
hypogammaglobulinemia, and a normal hematopoietic microenvironment. Dr.
Kazuo sugamura have been managed to produce II2rg (interleukin 2 receptor
subunit gamma) mutated mice (mutant mice have hypoplastic thymuses with
a 10-fold reduction in the absolute number of lymphocytes) by targeting the
gene in ES cells, and then injected to C57BL/6 (another strain of mice
commonly used as background strain for the generation of congenics carrying
both spontaneous and induced mutations) blastocysts.
Supplementary material
Supplementary material
Supplementary material
Supplementary material
Supplementary material
1. Pfcrt exporting the drugs via active transport
2. Pfmdr1 The gene located on chromosome 5, encodes ATP-binding cassette
(ABC) protein of 1419 amino acids and 162 (kDa) and thought to be an importer
of solutes into food vacuole as well as exporter of ATP-dependent of hidrophopic
compound from vacuole, including certain known drugs such as mefloquine,
halofantrine and the unrelated artemisinin derivatives.
3. Pfmrp Similarly to PfMDR1, PfMRP also belongs to family of ATP-binding
cassette (ABC) proteins and located on chromosome 1. It is an essential active
transporter involved in drug resistance that is indeed resided at the plasma
membrane (rather than food vacuole).
4. Pfnhe -
5. Folate pathway is an essential component for all living organisms to supply
constantly a fully reduced (tetrahydro) forms of folate for vital reactions of one-
carbon transfer, including determination of nucleotides for synthesizing DNA,
regardless endogenous or exogenous form of folate.
6. Plasmepsin II -
7. K13
Supplementary material

There are four major hypothesis of artemisinin resistance currently described. (1) elevated PIP3 production; initially, the
PfPI3K wild-type parasite, during exposure of artemisnin, will undergo 48-linked ubiquitination and subsequent proteasome
degradation of PfPI3K caused by a binding of PfKelch13 to PfPI3K, on the contrary, resistant-lines will be bindless and no
such degradation occure. These mechanism of PIP3 hypotesized to have a contribution to cell survival signaling pathway (
Davis, Lehmann et al. 2015; Mbengue, Bhattacharjee et al. 2015). (2) Up-regulation of ubiquitin/proteasome system; generally,
inhibition of protein translation followed by protein degradadions is particularly regulated by the ubiquitin/proteasome system
(Ron and Walter 2007; Amm, Sommer et al. 2014). Up-regulated ubiquitin/proteasome system was occurred in the artemisinin-
resistant parasites rather than susceptible one (Mok, Imwong et al. 2011; Dogovski, Xie et al. 2015). (3) Activation of unfolded
protein response pathway; most up-regulated genes in the resistant strain can export proteins, these up-regulation was
associated with unfolded protein response to react the endoplasmic reticulum stress, defining a mitigation of potential protein
damage by artemisinin (Witkowski, Lelivre et al. 2010; Hetz, Chevet et al. 2015; Mok, Ashley et al. 2015). (4) Lower
production of damaged protein; the accumulation of damaged protein can be beyond of parasites capacity to maintain
proteostasis and lead to death. Therefore, growth retardation of resistant strain parasites in the ring stage could lead to less
activated artemisinin by less hemoglobin digestion thus damaged protein are under proteostasis and survival rate of parasite
can achive higher then susceptible one (Dogovski, Xie et al. 2015).
Supplementary material