www.thelancet.com/infection Published online September 11, 2013 http://dx.doi.org/10.1016/S1473-3099(13)70252-4 1
Novel phenotypic assays for the detection of artemisinin-
resistant Plasmodium falciparum malaria in Cambodia:
in-vitro and ex-vivo drug-response studies
Benoit Witkowski*, Chanaki Amaratunga*, Nimol Khim, Sokunthea Sreng, Pheaktra Chim, Saorin Kim, Pharath Lim, Sivanna Mao,
Chantha Sopha, Baramey Sam, Jennifer M Anderson, Socheat Duong, Char Meng Chuor, Walter R J Taylor, Seila Suon, Odile Mercereau-Puijalon,
Rick M Fairhurst, Didier Menard
Background Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin
monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin
resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can
identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate
the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an
ex-vivo RSA can identify artemisinin-resistant P falciparum infections.
Methods We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study
done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov, number NCT00341003) and used novel in-vitro
survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to
dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah
Vihear, and Ratanakiri, Cambodia (NCT01736319), to measure the ex-vivo responses of parasites from patients with
malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the
Spearman correlation test.
Findings In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections
diff ered signifi cantly when assays were done on 0–3 h ring-stage parasites (10·88% vs 0·23%; p=0·007). Ex-vivo
survival rates signifi cantly correlated with in-vivo parasite clearance half-lives (n=30, r=0·74, 95% CI 0·50–0·87;
Interpretation The in-vitro RSA of 0–3 h ring-stage parasites provides a platform for the molecular characterisation of
artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is
Funding Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.
After the WHO’s recommendation1 to use artemisinin-
based combination therapies (ACTs) for the treatment of
Plasmodium falciparum malaria, the burden of this
disease declined substantially.2
antimalarial drugs,3 parasite resistance to artemisinin
and its derivatives has emerged in southeast Asia. Since
the fi rst reports in 2008 from Battambang4 province and
2009 from Pailin5 province, both in western Cambodia,
artemisinin-resistant P falciparum malaria has been
reported elsewhere in western Cambodia,6,7 western
Thailand,8 southern Burma,9 and southern Vietnam.10
Artemisinin resistance threatens malaria control,
treatment, and elimination eff orts worldwide.11,12 To
prevent the spread of artemisinin-resistant parasites
throughout southeast Asia and to Africa, rapid detection
of new artemisinin resistance foci and implementation
of containment interventions are a top priority.13
Although artemisinin resistance has not been
precisely defi ned, it is recognised as a relatively slow
As with earlier
parasite clearance rate in patients receiving an
artemisinin or ACT.14 The parasite clearance half-life can
be estimated from frequent parasite density counts in
patients with initial parasite densities of 10 000 parasites
per μL of blood or greater (ie, ≥0·2% parasitaemia).15 In
regions of low malaria transmission, like Cambodia,
parasite clearance studies require screening of
thousands of febrile individuals over entire transmission
seasons to enrol the few patients (<5%) who meet
inclusion criteria and agree to several days of
hospitalisation. Such studies are thus logistically and
fi nancially demanding, as well as inconvenient for
patients and their families. There is therefore an urgent
need to develop in-vitro and ex-vivo assay readouts that
correlate with parasite clearance half-life.
In-vitro readouts (ie, those obtained from culture-
adapted parasite lines in the laboratory) might be useful in
elucidating the molecular basis of artemisinin resistance
by providing robust phenotypes for genome-wide
association studies or the experimental validation of
September 11, 2013
*Joint fi rst authors
Malaria Molecular Epidemiology
Unit, Institut Pasteur du
Cambodge, Phnom Penh,
Cambodia (B Witkowski PhD,
N Khim MSc, P Chim,
S Kim, P Lim MD, D Menard PhD);
Laboratory of Malaria and
Vector Research, National
Institute of Allergy and
Infectious Diseases, National
Institutes of Health, Bethesda,
MD, USA (C Amaratunga PhD,
P Lim, J M Anderson PhD,
R M Fairhurst MD); National
Centre for Parasitology,
Entomology and Malaria
Control, Phnom Penh,
Cambodia (S Sreng, P Lim,
S Suon MD, Prof S Duong MD,
C M Chuor MD); Sampov Meas
Referral Hospital, Pursat,
Cambodia (S Mao MD); Makara
16 Referral Hospital, Preah
Vihear, Cambodia (C Sopha MD);
Ratanakiri Referral Hospital,
(B Sam MD); Service de
Médecine Internationale et
Universitaires de Genève,
(W R J Taylor MD); and Parasite
Molecular Immunology Unit,
Institut Pasteur, Paris, France
(O Mercereau-Puijalon PhD)
Dr Didier Menard, Institut
Pasteur du Cambodge,
5 Boulevard Monivong—BP 983,
Phnom Penh, Cambodia
Dr Rick M Fairhurst, National
Institutes of Health, 12735
Twinbrook Parkway, Room
3E-10A, Rockville, MD 20852, USA
www.thelancet.com/infection Published online September 11, 2013 http://dx.doi.org/10.1016/S1473-3099(13)70252-4
candidate molecular markers. Ex-vivo readouts (ie, those
obtained from uncultured parasite isolates collected
directly from patients in the fi eld) might be useful in
mapping the geographical spread or worsening of
artemisinin resistance in real-time, thus providing
actionable information for national malaria control
programmes. So far, consistent and signifi cant correlations
between half-lives and readouts from any in-vitro or ex-vivo
artemisinin susceptibility assay (eg, elevated IC50 value—
the drug concentration that inhibits parasite growth by
50%) have not been shown.4–6 One potential reason for this
observation is that parasites in these assays are exposed to
very low concentrations of dihydroartemisinin (the active
metabolite of all artemisinins) for 48–72 h, whereas
parasites in vivo are exposed to much higher concentrations
of dihydroartemisinin for only 1–2 h.
Artemisinin resistance in drug-selected P falciparum
lines has been associated with decreased susceptibility of
ring-stage parasites16–18 and, in some lines, mature
trophozoite-stage parasites as well.16,19 Using a novel in-
vitro assay (ring-stage survival assay; RSA),20 we recently
measured the susceptibility of 0–12 h post-invasion rings
to a pharmacologically relevant exposure (700 nM for 6 h)
to dihydroartemisinin. We noted a 17-times higher survival
rate of culture-adapted parasite isolates from Pailin
province, a region of artemisinin resistance in western
Cambodia, compared with those from Ratanakiri province,
a region of artemisinin sensitivity in eastern Cambodia.
We do not know how this geographical dichotomy relates
to the clinical artemisinin resistance phenotype, because
ring-stage parasites from patients with known parasite
clearance kinetics have not yet been tested in the RSA.
We aimed to assess whether an in-vitro RSA can
distinguish culture-adapted P falciparum isolates from
patients with slow-clearing or fast-clearing infections, to
investigate the stage-dependent susceptibility of parasites
to dihydroartemisinin in the in-vitro RSA, and to assess
whether an ex-vivo RSA can identify artemisinin-resistant
P falciparum infections in patients with malaria. To mimic
the in-vivo exposure of circulating, ring-stage parasites to
pharmacologically relevant doses of dihydroartemisinin,
we exposed synchronised, ring-stage parasites to brief,
high-dose pulses of this drug in the in-vitro RSA. We
similarly exposed ring-stage parasites obtained directly
from patients in the ex-vivo RSA.
Study design, patients, and drug treatment
We did two clinical studies in Cambodia to measure
therapeutic responses to artesunate. One study was done
in 2009–10 in Pursat province (western Cambodia;
registered with ClinicalTrials.gov, number NCT00341003),6
where artemisinins have been used for 35 years and
artemisinin resistance is well established, and the other in
2012 in Pursat province and also in Preah Vihear (northern
Cambodia) and Ratanakiri provinces (eastern Cambodia;
registered with ClinicalTrials.gov, number NCT01736319),
where ACTs were fi rst used in 2000 and artemisinin
resistance has not yet been reported. The studies were
done in referral hospitals in each province. The Cambodian
National Ethics Committee for Health Research and the
US National Institute of Allergy and Infectious Diseases
Institutional Review Board approved both studies.
The 2009–10 study in Pursat was previously reported.6
Patients were treated with oral doses of 4 mg/kg
artesunate at 0 h, 24 h, and 48 h, and then 15 mg/kg
mefl oquine at 72 h and 10 mg/kg mefl oquine at 96 h.
In the 2012 study, children older than 1 year and non-
pregnant adults with uncomplicated falciparum malaria
(parasite density ≥10 000 and ≤200 000 parasites per μL of
blood) were enrolled if written informed consent was
obtained from patients or parents or guardians of
children. Patients with severe malaria, Plasmodium vivax
infection, haematocrit less than 25%, antimalarial drug
use in the past 7 days, or known allergy to artemisinins
or piperaquine were excluded. Patients were treated with
oral doses of Duo-Cotecxin (containing 40 mg
dihydroartemisinin and 320 mg piperaquine per tablet;
Holleypharma, China) at 0 h, 24 h, and 48 h. The doses
were based on bodyweight: half a tablet (<10 kg), one
tablet (10–19 kg), one and a half tablets (20–29 kg), two
tablets (30–39 kg), and three tablets (≥40 kg).
Parasite density count, staging, and clearance
In the 2009–10 study, thick blood fi lms were made from
samples before the fi rst dose of artesunate (0 h) and then
every 6 h until asexual parasitaemia was undetectable.6 In
the 2012 study, blood fi lms were made at 0 h, 2 h, 4 h, 6 h,
8 h, and 12 h, and then every 6 h until parasitaemia was
undetectable. Parasite developmental stages at 0 h were
estimated as tiny or large rings on the basis of
morphological criteria (appendix). After patients
completed the study, parasite clearance curves were
derived from parasite density counts. The parasite
clearance half-life (ie, the time for parasite density to
decrease by 50%) was calculated from the slope constant
with the parasite clearance estimator.15,21 The half-life was
deemed interpretable when the R² value of the slope
regression line was greater than 0·8.
In-vitro parasite adaptation
In the 2009–10 study, blood samples were collected into
Franklin Lakes, NJ, USA) at 0 h. Parasitised erythrocytes
were cryopreserved in Glycerolyte 57 (Baxter Healthcare
Corp, Deerfi eld, IL, USA)22 immediately or after short-
term cultivation, and stored in liquid nitrogen until use.
Isotopic in-vitro sensitivity testing
The in-vitro sensitivity of culture-adapted parasites to
artesunate and dihydroartemisinin (obtained from the
Worldwide Antimalarial Resistance Network) was assessed
with a 48 h isotopic test20 with drug concen trations ranging
from 0·1 nM to 102·4 nM for artesunate, and from
See Online for appendix
www.thelancet.com/infection Published online September 11, 2013 http://dx.doi.org/10.1016/S1473-3099(13)70252-4 3
0·0625 nM to 64 nM for dihydroartemisinin. The quality
of in-vitro assays was monitored with the P falciparum 3D7
line. Results were expressed as the inhibitory
concentrations IC50 and IC90, defi ned as the drug
concentrations at which 50% or 90% of ³H-hypoxanthine
(Amersham, Les Ulis, France) incorporation was inhibited
compared with drug-free controls. IC50 and IC90 values were
established by non-linear regression with ICEstimator
In-vitro survival assays
Culture-adapted parasites were synchronised twice with
5% sorbitol (Sigma-Aldrich, Singapore) at 40 h intervals.
Synchronous 10–12 nuclei schizonts were incubated for
15 min at 37°C in RPMI-1640 supplemented with
15 U/mL of sodium heparin (Rotexmedica, Luitre,
France) to disrupt agglutinated erythrocytes, purifi ed on
a 35%/75% Percoll (Sigma-Aldrich) discontinuous
gradient, washed in RPMI-1640, and cultured for 3 h with
fresh erythrocytes. Cultures were treated with 5% sorbitol
to eliminate remaining schizonts, adjusted to 2%
haematocrit and 1% parasitaemia by adding uninfected
erythrocytes, and dispensed (2 mL per well in a 24-well
culture plate) into two parallel cultures. The RSA⁰–³ h was
done immediately with 0–3 h postinvasion rings, the
RSA⁹–¹² h with 9–12 h postinvasion rings, and the
trophozoite-stage survival assay (TSA¹⁸–²¹ h) with 18–21 h
In each assay, parasites were exposed to 700 nM
dihydroartemisinin or 0·1% dimethyl sulfoxide for 6 h,
washed with 12 mL RPMI-1640 to remove drug,
resuspended in complete medium (RPMI-1640, 0·5%
Albumax II, 2% heat-inactivated B+ plasma, 50 μg/mL
gentamicin), and cultured at 37°C in a tri-gas atmosphere
(5% CO2, 5% O2, 90% N2). Thin blood smears were
prepared and stained with 10% Giemsa (Merck KGaA,
Darmstadt, Germany) for 20 min. Survival rates were
assessed microscopically by counting the proportion of
viable parasites that developed into second-generation
rings or trophozoites with normal morphology at 66 h
(RSA⁰–³ h), 57 h (RSA⁹–¹² h), and 48 h (TSA¹⁸–²¹ h) after drug
removal. For each sample, roughly 10 000 erythrocytes
were assessed independently by two microscopists (BW
and CA) from whom each other’s data and half-lives were
masked. When the diff erence between survival rates was
greater than 20%, a third microscopist (DM), from whom
the data were also masked, assessed the slides.25 Mean
parasite counts were calculated and survival rates
expressed as ratios of viable parasitaemias in
dihydroartemisinin-exposed and dimethyl sulfoxide-
Ex-vivo survival assay
In the prospective 2012 study, ex-vivo RSAs were done on
parasites directly from consecutively enrolled patients in
Pursat, Preah Vihear, and Ratanakiri. 2 mL of venous
blood were collected into acid-citrate-dextrose vacutainers
before the fi rst Duo-Cotecxin dose and processed within
24 h. Plasma was removed and the blood washed three
times in RPMI-1640. If the parasitaemia was greater than
1%, it was adjusted to 1% by adding uninfected
erythrocytes. Ex-vivo RSAs were done as above except
that complete medium did not contain human plasma,
parasites were not experimentally synchronised, and
three diff erent atmospheres were tested in parallel: tri-
gas, 5% CO2, and candle jar. These atmospheres were
used to assess whether ex-vivo RSAs can produce
interpretable results in fi eld-based or under-resourced
settings where gas cylinders and gas-mixing incubators
might not be available or aff ordable. Smears made 66 h
after drug removal were assessed and survival rates
calculated as described above. Results were viewed as
interpretable if the parasitaemia in the sample exposed to
dimethyl sulfoxide was higher than the starting
DNA was extracted from 200 μL of whole blood collected
in 2010 at 0 h and from corresponding culture-adapted
parasites just before in-vitro assays using a QIAamp DNA
Blood Mini Kit (Qiagen, Valencia, CA, USA). Parasite
genotyping was done as described.26 12 single-nucleotide
polymorphisms were assessed with a PCR ligase detection
reaction fl uorescence microspheres assay (appendix).
For more on the ICEstimator see
Age range of parasites (h)
Time in culture (h)
Figure 1: Dihydroartemisinin survival assays
Synchronisation and timing of DHA exposure (A) for four in-vitro survival assays—RSA, previously described by
Witkowski and colleagues,20 RSA⁰?³ h, RSA⁹?¹² h, and TSA¹⁸?²¹ h—done on culture-adapted Plasmodium falciparum
isolates. During their 48 h cycle of intraerythrocytic development, parasites circulate as ring-stages (0–18 h) and
then sequester by specifi cally adhering to the endothelium of microvessels, where they mature into trophozoites
(18–36 h) and schizonts (36–48 h). Because of sequestration, clinical studies assess the clearance rate of circulating
ring-stage parasites only. In individual patients, the actual age-distribution of parasites circulating in peripheral
blood is unknown and can vary from patient to patient. The timing of dihydroartemisinin exposure for the ex-vivo
survival assay (B) done on circulating, ring-stage parasites (0–18 h) obtained directly from the blood of patients
with uncomplicated malaria. This assay thus measures the dihydroartemisinin susceptibility of the parasite isolate
at the same developmental stage and at the same time as the in-vivo parasite clearance study.
DHA=dihydroartemisnin. RSA=ring-stage survival assay. TSA=trophozoite-stage survival assay.
www.thelancet.com/infection Published online September 11, 2013 http://dx.doi.org/10.1016/S1473-3099(13)70252-4
Data were analysed with Microsoft Excel and MedCalc
version 12 (Mariakerke, Belgium). Quantitative data
were expressed as median (IQR). Stage-dependent
patterns of survival were expressed as the diff erence
between RSA⁰–³ h and TSA¹⁸–²¹ h (Δ). Continuous
variables were compared with the Mann-Whitney U
test. Correlations were analysed with the Spearman
correlation test. Ex-vivo RSA values that were obtained
in three atmospheric conditions were compared with
one-way repeated-measures ANOVA with Bonferroni
correction for p values (Friedman test). We deemed
signifi cant p values of less than 0·05.
Role of the funding source
The sponsors of the study had no role in study design,
data collection, data analysis, data interpretation, or
writing of the report. The corresponding authors had full
access to all the data in the study and had fi nal
responsibility for the decision to submit for publication.
From the 89 patients enrolled, we selected 18 fast-clearing
and 20 slow-clearing parasites representing the lower
and upper quartiles of the half-life distribution and
adapted them to culture as described.20 Assays were
ultimately done on parasites from 13 fast-clearing and
13 slow-clearing infections; the other 12 selected parasites
were excluded from the study because they did not adapt
to culture, did not have a corresponding half-life value
that was interpretable, or did not show an identical
genotype to the parasite originally obtained from the
Parasites were collected in Pursat in 2010 (appendix),
and used in three stage-specifi c survival assays (fi gure 1).
In the RSA for 0–3 h rings (RSA⁰–³ h), the median survival
rate of slow-clearing parasites was 47 times greater than
that of fast-clearing parasites (table, fi gure 2). By contrast,
9–12 h rings and 18–21 h trophozoites from fast-clearing
and slow-clearing infections showed no signifi cant
diff erence in survival (table, fi gure 2). The stage-
dependent survival patterns diff ered between fast-
clearing and slow-clearing parasites (fi gure 2, appendix).
Specifi cally, the survival rates of slow-clearing parasites
decreased with parasite stage, whereas those of fast-
clearing parasites increased with parasite stage (Δ=9·9%
[IQR 1·7 to 14·4] vs –0·3% [–1·1 to 0·4]; p=0·007). In an
isotope-based sensitivity assay that monitored replication
of parasites exposed to drug for 48 h,27 fast-clearing and
slow-clearing parasites did not diff er signifi cantly in IC50
and IC90 values for artesunate or dihydroartemisinin
In patients with falciparum malaria, the age
distribution of circulating ring-stage parasites is
heterogeneous, ranging from 0 h to 18 h at the time of
clinical presentation;28 that is, ring-stage parasites are not
necessarily tightly synchronised at the 0–3 h age of
development. We therefore sought to assess whether an
ex-vivo RSA could distinguish fast-clearing from slow-
clearing parasites that have been neither culture-adapted
nor experimentally synchronised.
In the prospective 2012 study, 30 (83%) of 36 patients
had interpretable half-life values and tri-gas survival rates
(appendix), which correlated signifi cantly (fi gure 3).
Parasite survival rates did not diff er between the three
atmospheres (n=26; p=0·30, Friedman test). The ex-vivo
RSA accurately identifi ed artemisinin-resistant infections
where they have not been previously described (fi gure 3,
appendix). In Preah Vihear, for example, one parasite
with a 12·2% survival rate had an 8·17 h half-life, whereas
the other six parasites with a median 0·70% survival rate
0·23 (0·14–2·93, 0·01–51·39)
1·07 (0·77–1·70, 0·06–10·00)
0·99 (0·48–2·20, 0·16–4·10)
0·71 (0·58–0·94, 0·29–1·20)
2·60 (2·28–3·30, 1·54–4·49)
1·00 (0·84–1·47, 0·28–1·71)
3·32 (2·52–3·94, 2·30–5·80)
10·88 (4·75–13·91, 0·16–29·14)
2·12 (1·46–3·55, 0·33–8·00)
1·16 (0·78–2·05, 0·38–5·30)
0·79 (0·62–0·11, 0·42–1·51)
2·46 (1·78–3·02, 1·48–4·40)
1·11 (0·98–1·84, 0·83–2·50)
3·02 (2·38–3·86, 1·99–6·38)
Data are median (IQR, range). Percentage survival in RSA⁰?³ h, RSA⁹?¹² h, and TSA¹⁸?²¹ h, and IC50 and IC90 values for
dihydroartemisinin and artesunate in isotope-based sensitivity assays. p values for signifi cance from Mann-Whitney
test. RSA=ring-stage survival assay. TSA=trophozoite-stage survival assay.
Table: Parasite survival in in-vitro assays using culture-adapted isolates
Figure 2: In-vitro survival after exposure to dihydroartemisinin
Results are expressed as the proportion of viable Plasmodium falciparum parasites after a 6 h exposure of 0–3 h
rings (RSA⁰?³ h), 9–12 h rings (RSA⁹?¹² h), and 18–21 h trophozoites (TSA¹⁸?²¹ h) to 700 nM dihydroartemisinin
compared with dimethyl sulfoxide. These assays were done on culture-adapted parasite isolates obtained from
13 patients with fast-clearing infections (fi lled circles) and 13 patients with slow-clearing infections (open circles)
in Pursat in 2010. The horizontal lines represent the medians and whiskers the IQRs. The solid lines show stage-
dependent survival pattern of parasites from slow-clearing infections and the dotted lines the stage-dependent
survival pattern of parasites from fast-clearing infections. RSA=ring-stage survival assay. TSA=trophozoite-stage
Proportion of viable parasites (%; dihydroartemisinin-exposed/non-exposed)
www.thelancet.com/infection Published online September 11, 2013 http://dx.doi.org/10.1016/S1473-3099(13)70252-4 5
(IQR 0·18–2·0) had a median 2·28 h half-life (1·89–3·52).
In Ratanakiri, one parasite with a 38·3% survival rate
had a 9·06 h half-life, whereas the other ten parasites had
a median 0·40% survival rate (0·26–1·48) and a median
2·28 h half-life (1·90–2·64). Our fi ndings suggest that
artemisinin-resistant P falciparum has spread or
independently emerged in northern and eastern
Cambodia, a possibility that can now be confi rmed with
the in-vitro RSA⁰–³ h.
P falciparum isolates from slow-clearing and fast-clearing
infections in Cambodia respond diff erently to a 6 h,
700 nM exposure to dihydroartemisinin. In the RSA⁰–³ h,
rings of slow-clearing parasites had much higher survival
rates than those of fast-clearing parasites. In the ex-vivo
RSA, survival rates correlated with parasite clearance half-
lives. Importantly, the ex-vivo RSA accurately identifi ed
slow-clearing infections in Cambodian provinces where
they have not yet been described. To our knowledge, these
are the fi rst reported in-vitro and ex-vivo dihydro-
artemisinin susceptibility data that correlate with in-vivo
parasite clearance half-lives (panel). These data qualify the
in-vitro RSA⁰–³ h as a new laboratory test for elucidating the
mechanism of artemisinin resistance through molecular
studies. These studies might include genome-wide
association studies,29 associating RSA⁰–³ h survival rates
with whole-genome single-nucleotide polymorphism
data, phenotypic screening of parasite progeny clones
obtained from genetic crosses between artemisinin-
sensitive and artemisinin-resistant parental lines,
phenotypic characterisation of the diff erent artemisinin-
resistant parasite subpopulations circulating in western
Cambodia,29 and validation of candidate molecular
markers through genetic manipulation of parasites.
Our fi ndings also suggest that the ex-vivo RSA is a
feasible, convenient method for detecting the spread and
emergence of artemisinin resistance in areas where it
has not yet been reported (eg, eastern Cambodia), or the
worsening of artemisinin resistance where it is
entrenched (eg, western Cambodia). Both types of
fi ndings from ex-vivo RSAs might inform national
malaria control programmes to expand or intensify
containment measures. In a screen and confi rm
approach to support such eff orts, we propose that the ex-
vivo RSA be used in the fi eld to screen for artemisinin-
resistant parasites. Any parasite showing dihydro-
artemisinin resistance in this assay can then be adapted
to short-term culture in a laboratory, genotyped to ensure
its identity to the clinical parasite isolate obtained from a
patient, and tested to confi rm dihydroartemisinin
resistance with the in-vitro RSA⁰–³ h.
For both artemisinin-sensitive and artemisinin-
resistant parasites, we show stage-dependent hetero-
geneity of dihydroartemisinin susceptibility in ring
forms. In artemisinin-sensitive parasites, 0–3 h rings
were more susceptible to dihydroartemisinin than were
9–12 h rings. This fi nding with clinical parasite isolates is
consistent with the recent report that 2–4 h rings of
artemisinin-sensitive laboratory lines are specifi cally
hypersensitive to dihydroartemisinin.18 However, in
artemisinin-resistant parasites, 0–3 h rings were less
susceptible to dihydroartemisinin than were 9–12 h rings.
We tentatively conclude that the susceptibility of
Cambodian parasites to dihydroartemisinin is controlled
predominantly at the 0–3 h stage of parasite development.
This interpretation, and our fi nding that trophozoites are
mostly susceptible to dihydroartemisinin irrespective of
half-life, is consistent with mathematical modelling
predictions30 and transcriptomics data31 from studies of
Half-lives and RSA⁰–³ h survival rates were discordant in
four patients (fi gure 2, appendix). Three patients (1007,
1006, and 1009) had fast-clearing infections with parasites
showing survival rates of 5·3%, 19·3%, and 51·4%, and a
resistant stage-dependent pattern (Δ=1·2%, 17·3%, and
50·2%, respectively). Their patterns diff ered from those
of fast clearing-infections (Δ=–0·7% vs 17·3%; p=0·01),
being similar to those from slow-clearing-infections
(Δ=10·3% vs 17·3%; p=0·56; appendix). To explain this
discordance, we postulated that these three parasites had
already developed into dihydroartemisinin-susceptible,
late ring-stage parasites in the patients’ blood at the time
of the fi rst artesunate dose.
To assess this possibility, we reviewed the initial blood
smears from these patients and estimated the relative
Figure 3: Correlation of in-vivo parasite clearance half-lives and ex-vivo dihydroartemisinin survival rates
Ex-vivo ring-stage survival assays (RSAs) were done on parasite isolates obtained directly from patients with
malaria in Pursat, Preah Vihear, and Ratanakiri in 2012. Results from the ex-vivo RSAs are expressed as the
proportion of viable parasites after a 6 h exposure to 700 nM dihydroartemisinin compared with dimethyl-
sulfoxide-exposed controls. Results from the parasite clearance studies are expressed as the parasite clearance
half-life in hours. The proportion of viable parasites in ex-vivo RSAs correlated signifi cantly with the parasite
clearance half-life (r=0·74, 95% CI 0·50–0·87; p<0·0001) in Pursat (red), Preah Vihear (blue), and Ratanakiri (green).
Proportion of viable parasites (%; dihydroartemisinin-exposed/non-exposed)
Parasite clearance half-life (h)
www.thelancet.com/infection Published online September 11, 2013 http://dx.doi.org/10.1016/S1473-3099(13)70252-4
age of their ring-stage parasites just before treatment
with artesunate (appendix). In thin blood smears made at
0 h, we noted that these three discordant patients indeed
had a two-times lower proportion of tiny rings compared
with the 12 concordant patients from the slow-clearing
group (ie, those having slow-clearing infections with
dihydroartemisinin-resistant parasites; 42·4% vs 75·0%;
p=0·03). Higher proportions of large, older rings could
account for shorter than expected half-lives because these
forms are more susceptible to dihydroartemisinin than
tiny, young rings. Overall, our fi ndings suggest that the
relative abundance of tiny and large rings at the time of
the fi rst artemisinin dose aff ects the parasite clearance
half-life, and that accurate ex-vivo staging of parasites is
crucial for classifying treatment outcome. In one patient
(896), having a slow-clearing infection with a parasite
showing a survival rate of 0·2%, and a sensitive stage-
dependent pattern (Δ=–1·4%; appendix), we cannot rule
out an inadequate immune response to infection32 or
insuffi cient plasma concentrations of artemisinins.
The RSA⁰–³ h survival rate might be crucially informative
in ongoing parasite genetics studies31,33,34 aimed at
identifying loci under artemisinin selection, because it is
unaff ected by in-vivo variables (eg, pharmacokinetics,
haemoglobin type, and acquired immunity) that might
aff ect the parasite clearance half-life. Although this
phenotype might also be a useful readout in studies to
defi ne and validate molecular markers for tracking
artemisinin-resistant parasites in the fi eld, the RSA⁰–³ h is
a laborious assay. By contrast, the ex-vivo RSA saves
weeks of eff ort (results are available in 3 days), and avoids
the confounding eff ects of parasite clone elimination and
metabolic changes that might accompany the culture
adaptation of parasites. In addition to implementing
methods that more precisely establish the age of rings,
FACS-based or ELISA-based analysis of parasite viability
should improve the throughput of dihydroartemisinin-
susceptibility studies. Until such methods are developed
and validated, we propose the simple ex-vivo RSA as a
highly informative surveillance approach for the
identifi cation of artemisinin-resistant parasites in areas
where slow parasite clearance is suspected. Investigating
the relation between RSA survival rates ex vivo and
parasite recrudescence rates in vivo might be useful in
assessing the clinical eff ect of artemisinin resistance.
BW, CA, PL, JMA, SK, SD, CMC, WRJT, OM-P, RMF, and DM
contributed to study design. NK genotyped parasites. BW, CA, and PC
did the in-vitro and ex-vivo drug assays. SSr, SM, CS, BS, and SSu
gathered clinical data. BW, CA, OM-P, RMF, and DM analysed data and
wrote the report.
Confl icts of interest
We declare that we have no confl icts of interest.
We thank Robert Gwadz, Savuth Koeuth, François Nosten, Eng Ly Pech,
Thomas Wellems, and Chongjun Zhou for their eff orts in support of
this work. This study was funded by the Intramural Research Program,
NIAID, NIH, and by grants from Institut Pasteur du Cambodge (Institut
Pasteur, International Division and Banque Natixis) and Laboratoire
d’excellence IBEID (Agence Nationale de la Recherche, France). BW is
supported by a postdoctoral fellowship from the International Division,
Institut Pasteur, and DM by the French Ministry of Foreign Aff airs.
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Plasmodium falciparum in Pursat province, western Cambodia:
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7 Amaratunga C, Mao S, Sreng S, et al. Slow parasite clearance rates
in response to artemether in patients with severe malaria.
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Panel: Research in context
We searched PubMed with the terms “artemisinin resistant malaria”, limited our search to
clinical trials, and used no date or language restrictions. This process produced
56 publications. Any in-vitro or ex-vivo drug assays done in these studies involved the
continuous exposure of Plasmodium falciparum parasites to very low concentrations of
artemisinins during the entire lifecycle of their blood-stage development. Results of these
assays have not consistently correlated with clinical effi cacy.4–6 Only four recent publications
describe new assays that were specifi cally designed to measure P falciparum susceptibility to
artemisinins.17–20 Klonis and colleagues18 described an assay working with laboratory-adapted
parasites that were artemisinin-sensitive. Witkowski and coworkers17 and Teuscher and
colleagues19 described assays working with laboratory-adapted parasites and their
drug-selected counterparts that became resistant to artemisinins. Witkowski and colleagues20
described a more relevant study using parasites from western and eastern Cambodia, where
parasites are commonly resistant and sensitive to artemisinin respectively. None of these
studies were designed to test whether in-vitro susceptibility data correlated with in-vivo
effi cacy data (ie, parasite clearance rates after artemisinin treatment, the currently accepted
clinical phenotype); therefore, these assays could not be clinically validated.
We report for the fi rst time novel in-vitro and ex-vivo ring-stage survival assays (RSAs) that
detect artemisinin-resistant, slow-clearing P falciparum infections in patients with malaria.
In both assays, early ring-stage parasites are exposed to a pharmacologically relevant pulse
of dihydroartemisinin and their survival measured 72 h later. With parasites adapted to
culture in the laboratory, the in-vitro RSA can be used to discover the molecular
mechanisms of artemisinin resistance, to investigate the mode of action of artemisinins,
and to identify artemisinin-resistant parasite strains for testing next-generation
antimalarial drugs. The ex-vivo RSA with parasites obtained directly from patients with
malaria can be easily implemented in fi eld-based settings to monitor the worsening of
artemisinin resistance where it is highly prevalent (eg, western Cambodia), and to map its
spread or independent emergence elsewhere in the Greater Mekong Subregion. Also, this
simple test might readily be established at sentinel sites in sub-Saharan Africa, where the
arrival of artemisinin-resistant P falciparum is expected to be especially devastating. The
ex-vivo RSA can thus provide crucial surveillance data to the national malaria control
programmes of all countries threatened by artemisinin-resistant malaria.
For the standard operating
procedures for ex-vivo and
in-vitro RSA see http://www.
www.thelancet.com/infection Published online September 11, 2013 http://dx.doi.org/10.1016/S1473-3099(13)70252-4 7
9 Kyaw MP, Nyunt MH, Chit K, et al. Reduced susceptibility of
Plasmodium falciparum to artesunate in southern Myanmar.
PLoS One 2013; 8: e57689.
10 Hien TT, Thuy-Nhien NT, Phu NH, et al. In vivo susceptibility of
Plasmodium falciparum to artesunate in Binh Phuoc Province,
Vietnam. Malar J 2012; 11: 355.
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artemisinin-resistant malaria. N Engl J Med 2011; 365: 1073–75.
13 WHO. Emergency response to artemisinin resistance in the Greater
Mekong subregion: regional framework for action 2013–2015.
Geneva: World Health Organization, 2013.
14 White NJ. The parasite clearance curve. Malar J 2011; 10: 278.
15 Flegg JA, Guerin PJ, White NJ, et al. Standardizing the
measurement of parasite clearance in falciparum malaria: the
parasite clearance estimator. Malar J 2011; 10: 339.
16 Cui L, Wang Z, Miao J, et al. Mechanisms of in vitro resistance to
dihydroartemisinin in Plasmodium falciparum. Mol Microbiol 2012;
17 Witkowski B, Lelievre J, Barragan MJ, et al. Increased tolerance to
artemisinin in Plasmodium falciparum is mediated by a quiescence
mechanism. Antimicrob Agents Chemother 2010; 54: 1872–77.
18 Klonis N, Xie SC, McCaw JM, et al. Altered temporal response of
malaria parasites determines diff erential sensitivity to artemisinin.
Proc Natl Acad Sci USA 2013; 110: 5157–62.
19 Teuscher F, Chen N, Kyle DE, et al. Phenotypic changes in
artemisinin-resistant Plasmodium falciparum lines in vitro: evidence
for decreased sensitivity to dormancy and growth inhibition.
Antimicrob Agents Chemother 2012; 56: 428–31.
20 Witkowski B, Khim N, Chim P, et al. Reduced artemisinin
susceptibility of Plasmodium falciparum ring stages in western
Cambodia. Antimicrob Agents Chemother 2012; 57: 914–23.
21 WWARN. Parasite clearance estimator. https://www.wwarn.org/
Aug 21, 2013).
22 Moll K, Ljungström I, Perlmann H, et al (eds). Methods in malaria
research, 5th edn (version 5.2 revision). http://www.mr4.org/
(accessed Aug 21, 2013).
23 Le Nagard H, Vincent C, Mentre F, et al. Online analysis of in vitro
resistance to antimalarial drugs through nonlinear regression.
Comput Methods Programs Biomed 2011; 104: 10–18.
24 Kaddouri H, Nakache S, Houze S, et al. Assessment of the drug
susceptibility of Plasmodium falciparum clinical isolates from Africa
by using a plasmodium lactate dehydrogenase immunodetection
assay and an inhibitory maximum eff ect model for precise
measurement of the 50-percent inhibitory concentration.
Antimicrob Agents Chemother 2006; 50: 3343–49.
25 WHO. Malaria microscopy quality assurance manual—version 1.
Geneva: World Health Organization, 2009.
26 Daniels R, Volkman SK, Milner DA, et al. A general SNP-based
molecular barcode for Plasmodium falciparum identifi cation and
tracking. Malar J 2008; 7: 223.
27 Desjardins RE, Canfi eld CJ, Haynes JD, et al. Quantitative
assessment of antimalarial activity in vitro by a semiautomated
microdilution technique. Antimicrob Agents Chemother 1979;
28 Silamut K, White NJ. Relation of the stage of parasite development
in the peripheral blood to prognosis in severe falciparum malaria.
Trans R Soc Trop Med Hyg 1993; 87: 436–43.
29 Miotto O, Almagro-Garcia J, Manske M, et al. Multiple populations
of artemisinin-resistant Plasmodium falciparum in Cambodia.
Nat Genet 2013; 45: 648–55.
30 Saralamba S, Pan-Ngum W, Maude RJ, et al. Intrahost modeling of
artemisinin resistance in Plasmodium falciparum.
Proc Natl Acad Sci USA 2010; 108: 397–402.
31 Mok S, Imwong M, Mackinnon MJ, et al. Artemisinin resistance in
Plasmodium falciparum is associated with an altered temporal
pattern of transcription. BMC Genomics 2011; 12: 391.
32 Lopera-Mesa TM, Doumbia S, Chiang S, et al. Plasmodium
falciparum clearance rates in response to artesunate in Malian
children with malaria: eff ect of acquired immunity. J Infect Dis 2013;
33 Cheeseman IH, Miller BA, Nair S, et al. A major genome region
underlying artemisinin resistance in malaria. Science 2012;
34 Takala-Harrison S, Clark TG, Jacob CG, et al. Genetic loci associated
with delayed clearance of Plasmodium falciparum following
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This webappendix formed part of the original submission and has been peer reviewed.
We post it as supplied by the authors.
Supplement to: Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays
for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia:
in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 2013; published online
Sept 11. http://dx.doi.org/10.1016/S1473-3099(13)70252-4.
Novel phenotypic assays detect artemisinin-resistant Plasmodium falciparum malaria in
Cambodia: in-vitro and ex-vivo drug response studies
Benoit Witkowski, Chanaki Amaratunga, Nimol Khim, Sokunthea Sreng, Pheaktra Chim, Saorin Kim, Pharath Lim,
Sivanna Mao, Chantha Sopha, Baramey Sam, Jennifer M. Anderson, Socheat Duong, Char Meng Chuor, Walter R.
J. Taylor, Seila Suon, Odile Mercereau-Puijalon, Rick M. Fairhurst, Didier Menard
Appendix 1: Patient information and corresponding data from in-vitro assays performed on P falciparum isolates
from Pursat in 2010.
Appendix 2: Protocols, PCR/nested PCR primer sequences, and LDR probe sequences used to genotype P.
falciparum isolates obtained in Pursat in 2010.
Appendix 3: Patient information and corresponding data from ex-vivo assays performed on P falciparum isolates
from Pursat, Preah Vihear, and Ratanakiri in 2012.
Appendix 4: Grading of asexual P falciparum parasites into two developmental categories: ‘tiny’ (Panel A) and
‘large’ (Panel B) rings.
Appendix 5: Selection of P falciparum isolates from Pursat in 2010 for culture adaptation and use in in-vitro assays.
Appendix 6: Individual stage-dependent patterns in in-vitro survival assays (RSA0-3h, RSA9-12h, and TSA18-21h)
performed on parasite isolates from fast- (Panel A) and slow-clearing (Panel B) infections in Pursat in 2010.
Appendix 1: Patient information and corresponding data from in-vitro assays performed on P falciparum
isolates from Pursat in 2010.
Discordant samples are shown in bold.
0 hours (/mm3)
curve - R2
% of tiny
906 23 M 33,742 1·01 0·15 0·28 0·29 42·4
919 37 F 250,000 3·03 0·8847 0·01 0·06 0·17 0·77 0·58 82·9
970 23 M 100,936 3·59 0·8685 0·25 2·1 0·12 0·94 0·88 86·4
189-4 13 M 272,000 3·65 0·9660 0·23 0·46 0·50 1·69 0·97 76·8
915 18 M 50,633 3·69 0·9357 0·35 0·94 0·37 1·34 0·68 65·6
931 29 M 296,666 4·25 0·8884 0·56 1·07 0·52 0·82 0·57 85·4
911 19 M 51,576 4·46 0·8672 0·19 0·60 0·32 0·98 0·76 81·8
918 58 M 351,111 4·54 0·9377 0·14 0·97 0·14 1·52 0·90 77·4
1003 31 M 25,920 4·56 0·9839 0·05 1·16 0·04 1·00 0·60 37·5
1006 48 M 11,882 4·67 0·9680 19·32 6·27 3·08 1·33 0·71 64·8
1007 24 M 16,466 4·71 0·9237 5·30 1·30 4·08 1·71 1·20 42·4
1009 42 M 27,714 4·77 0·9314 51·39 10 5·14 0·87 0·40 27·0
945 10 M 188,500 4·83 0·8714 0·22 1·09 0·20 1·42 1·01 78·1
968 64 M 36,730 7·97 0·9855 8·34 1·71 4·88 0·96 0·68 57·5
818-2 46 M 47,835 7·97 0·9877 13·48 8·00 1·69 1·00 0·81 88·8
976 44 M 65,432 8·21 0·9305 2·18 0·78 2·79 1·14 0·79 65·0
946 17 M 53,626 8·26 0·9458 7·35 1·20 6·13 1·95 1·04 94·4
969 20 M 95,304 8·32 0·9599 6·30 2·12 2·98 2·50 1·51 75·0
950 15 F 79,714 8·54 0·.9495 3·20 3·48 0·92 1·71 1·30 81·8
958 30 M 41,553 8·73 0·9851 29·14 3·62 8·05 1·11 0·71 43·7
896 21 M 82,807 8·75 0·9322 0·16 0·33 0·48 0·83 0·42 76·4
955 48 M 20,242 9·05 0·9326 11·80 2·20 5·36 1·89 1·18 77·4
938 18 M 22,109 9·11 0·9655 14·33 4·00 3·58 0·85 0·49 57·0
990 31 M 18,125 9·45 0·9775 12·60 3·00 4·20 1·09 0·55 75·0
922 26 M 42,240 9·72 0·9589 21·90 2·10 10·43 1·80 0·95 74·5
956 20 M 48,000 10·08 0·9489 10·88 2·01 5·42 1·11 0·69 84·8
Appendix 2: Protocols, PCR/nested PCR primer sequences, and LDR probe sequences used to genotype P falciparum isolates obtained in Pursat in 2010.
Outer PCR primer sequences
Inner PCR primer sequences
SNP Upstream allele-specific probe sequence
Downstream conserved probe sequences
(with 5' phosphorylation and 3' biotinylation)
TGGAAATACACAATTCAATG TTCCAAAACTATGTTTGCTGCT C cacttaattcattctaaatctatcTTTCAAATGTTATTTTCAACTATGTTAAGTAAC
CGAATGTTTTTCCATATTTT TGCAGTGGTACTTGTTGCTACC T tactacttctataactcacttaaaTTTCAAATGTTATTTTCAACTATGTTAAGTAAT
CCAACCAACGAACACAAATAC AGGAAAATGCTCCGGTAACT T actacttattctcaaactctaataGAAAAAAATAATTTGAACAATAAAACTTATAATAA
TGGTTGACTGTTATTGGGGTA GGTTCATATTATTTGGTGACTCG C acttatttcttcactactatatcaGAAAAAAATAATTTGAACAATAAAACTTATAATAG
TGAATGTAATATAAATCAGGTTG CTGAAAAATCGGATGAATGG G cactacacatttatcataacaaatAAGGAGATAGTGTTGGGGGC
GGCTGGAATAGATAAAATCA GGCTAGCTCAGCTTCCAAT A aactttctctctctattcttatttAAGGAGATAGTGTTGGGGGT
CGAATTTAAGTACCTTAGGAAA TCACAACGTCCATATGTTGAA G tcatcactttctttactttacattTGATGAAAGCCACCGAACTC
TCATAAAGTTTTTATTGTCTTCA TCATTATCACCTACTTTCTGTACCA A tacacaatattcatcataactaacTGATGAAAGCCACCGAACTT
GAGGATGTATACCATTAGCTG GATGAGTTAGCAACGAAACCA T cataatcaatttcaactttctactCCATCATATAAATATTTCTATATTCCATTAGCT
ATCATTCATATGTGGAAACA AACGTAAACCAGGAGTAAGACG A caaatacataatcttacattcactCCATCATATAAATATTTCTATATTCCATTAGCA
12 ATACACTAAACGCAAAACCT CATTATGCGAATGCGATCTA G ctttctcatactttcaactaatttAATGGAAAATTTTGATGATATTTTATTAAG
TGTTAATTCCTTTTCGATTT CGTTTATATTGCAACATTTCTTCA A tcaaactctcaattcttacttaatAATGGAAAATTTTGATGATATTTTATTAAA
TGACAAACAAGTATATAATAATAAGAG TGTTGTTGGTGAATACAATGAAA G cttaacatttaacttctataacacAAATAACAATGAACATCATCATGATG
TGTTTTAAAAGTCGTGGATA TCGTACCACCATTAACATTTTG A tacaacatctcattaacatatacaAAATAACAATGAACATCATCATGATA
CATAAATAAAACTTTCGCTGA TGGAATGATTTGAGCAATAGAA C ttaaacaatctactattcaatcacAAATTCAAATTATGTTCACAGGAATAAAC
ATTTTCAATATCATCTTCTTTACA AATACCCATGATATCACATTCCA A tctctttaaacacattcaacaataAAATTCAAATTATGTTCACAGGAATAAAA
ATCATCTGTATTTTGTTATTATGA AATCTTTTCCAGTTATTTTCTATCCA C aatcaacacacaataacattcataACCTTCCATATCTAAAAAAACTTCATTC
GTTAGACAATTTTGCTACACTT CATGGGGGTATGTAATTTGG A caatttacatttcactttcttatcACCTTCCATATCTAAAAAAACTTCATTA
TCACAAACAAATAACAATGAA AAAAGCAATTCCACAAGAACC A ttcttcattaacttctaatcttacCCTACATTAAATGAAAATGAAAACGTTA
ACATGTTTTGGACCATCTAC CTGGTGTTTCCTTTTTATTTGG C ttaacaacttatacaaacacaaacCCTACATTAAATGAAAATGAAAACGTTC
AATATATCTGTATTTGCTAACATGA TGTGTTTTATTTTTAGTGTGAGCTTT C cataatcaatttcaactttctactCAAAATATCAACAAGAAAAACATAATTACTC
TGTAACAAGGAATGACAAAA AGAGGATATCCAATAGGGTGCT T caaatacataatcttacattcactCAAAATATCAACAAGAAAAACATAATTACTT
CGATTTAATTACTGTTTTGAGA AACAAATCATCAATTAAGTCATCC G cacttaattcattctaaatctatcAATTAGAAAATACACAAAATTATCAAAAAAG
TTGGTTTACAATTAGTTCTAGC TGAGGAATAGGTTCATATGCTG T tttacaaatctaatcacactatacAATTAGAAAATACACAAAATTATCAAAAAAT
First-round PCRs were performed in the following reaction mixture: 2·5 µL 10X buffer, 2·5 mM MgCl2, 0·2 mM each deoxynucleoside triphosphate, 0·25 µM each primer,
1·25 U FirePol Taq polymerase (Solis Biodyne, Tartu, Estonia), and 5 µL DNA template. Nested PCRs were performed in the same reaction mixture with 3 μL of first-round
PCR products (diluted 1:10) added. PCR amplifications were performed under the following conditions: first-round PCR - 95°C for 15 min and 30 cycles at 95°C for 30 s,
52°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 10 min; nested PCR - 95°C for 15 min and 40 cycles at 95°C for 10 s, 57°C for 15 s, 72°C for 20 s, and a final
extension at 72°C for 10 min. As previously described,1-3 a ligase detection reaction between modified upstream allele-specific (with unique 5' extremity TAG sequences) and
downstream conserved sequence primers (with a 5' phosphorylation and 3' biotinylation) were performed using 1 µL of nested PCR products in 15 μL solution of 20 mM Tris-
HCl buffer (pH 7·6), 25 mM potassium acetate, 10 mM magnesium acetate, 1 mM NAD+, 10 mM dithiothreitol, 0·1% Triton X-100, 10 nM each LDR probe, and 2 U of Taq
DNA ligase (New England Biolabs, Beverly, MA, USA). Reaction mixtures were heated to 95°C for 1 min, followed by 32 cycles at 95°C for 15 s and 60°C for 2 min. In a
second step, 5 µL of multiplex LDR products were added to 60 μL of hybridization solution (3 M tetramethylammonium chloride [TMAC], 50 mM Tris-HCl [pH 8·0], 3 mM
EDTA [pH 8·0], 0·10% sodium dodecyl sulfate) containing 2500 MagPlex-TAG Microspheres® (Luminex, Austin, TX, USA) for each allelic set, heated to 95°C for 90 s and
incubated at 37°C for 40 min to allow hybridization between SNP-specific LDR products and microsphere-labelled anti-TAG probes. Following hybridization, 6 μL of
streptavidin-R-phycoerythrin (Molecular Probes, Eugene, OR, USA) in TMAC hybridization solution (20 ng/μL) was added and incubated at 37°C for 40 min in Costar 6511
M polycarbonate 96-well V-bottom plates (Corning Inc., Corning, NY, USA). Detection of SNP-specific products was performed through a MagPix machine (Luminex).
Fluorescence data were managed by xPONENT software (Luminex) and entered into Microsoft Excel software (Microsoft Office 2010). In each run, samples were analyzed
with 3D7, Dd2, and HB3 genomic DNA controls and no template control.
1. Barnadas C, Kent D, Timinao L, et al. A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps
and mdr-1 genes. Malar J 2011;10:282.
2. Carnevale EP, Kouri D, DaRe JT, McNamara DT, Mueller I, Zimmerman PA. A multiplex ligase detection reaction-fluorescent microsphere assay for simultaneous
detection of single nucleotide polymorphisms associated with Plasmodium falciparum drug resistance. J Clin Microbiol 2007;45:752-61.
3. McNamara DT, Thomson JM, Kasehagen LJ, Zimmerman PA. Development of a multiplex PCR-ligase detection reaction assay for diagnosis of infection by the four
parasite species causing malaria in humans. J Clin Microbiol 2004;42:2403-10.
Appendix 3: Patient information and corresponding data from ex-vivo assays performed on P. falciparum
isolates from Pursat, Preah Vihear, and Ratanakiri in 2012.
at 0 hours
curve - R2
Ex-vivo RSA value (%)
tri-gas candle-jar 5%CO2
KH1, KH2, and KH3 are identifying codes for Pursat, Preah Vihear, and Ratanakiri, respectively; these codes are
not related to the parasite subpopulations reported by Miotto et al. (Nat Genet, 2013).
Appendix 4: Grading of asexual P falciparum parasites into two developmental categories: ‘tiny’ (Panel A)
and ‘large’ (Panel B) rings.
Representative photomicrographs of P. falciparum isolates collected from patients just prior to receiving a first dose
of artesunate. Giemsa-stained thin blood films are shown. Rings were classified as ‘tiny rings’ when the width of the
cytoplasm band was less than, or equal to, half of the diameter of the nucleus (Panel A) and as ‘large rings’ when the
width of the cytoplasm band was greater than the diameter of the nucleus (Panel B).
7 Download full-text
Appendix 5: Selection of P falciparum isolates from Pursat 2010 for culture adaptation and use in in-vitro
89 isolates collected from P. falciparum infected
patients in Pursat province, Cambodia, 2010
ISA: Isotope-based assay; RSA0-3h: Ring-stage survival assay with 0-3 hour rings; RSA9-12h: Ring-stage survival
assay with 9-12 hour rings & TSA18-21h: Trophozoite-stage survival assay with 18-21 hour trophozoites.
18 isolates selected for in-vitro assays
from the lower interquartile range of
half-life defined as fast-clearing
infections (half-life <4·5 hours)
14 isolates successfully adapted for
14 isolates with interpretable
parasite clearance half-lives
20 isolates selected for in-vitro assays
from the higher interquartile range of
half-life defined as slow-clearing
infections (half-life >7·5 hours)
15 isolates with interpretable
parasite clearance half-lives
17 isolates successfully adapted for
13 isolates with similar bar-coding
genotypes between parasites at 0 hours
and parasites post-culture adaptation
13 isolates with similar bar-coding
genotypes between parasites at 0 hours
and parasites post-culture adaptation
13 culture-adapted parasites from
slow-clearing infections tested in
ISA, RSA0-3h, RSA9-12h& TSA18-21h
0 2 4 6 8 10 12
1 2 3 4 5 6 7 8 9 10 11 12
Parasite clearance half-life (h)
IQR > 75%IQR < 25%
13 culture-adapted parasites from
fast-clearing infections tested in ISA,
RSA0-3h, RSA9-12h& TSA18-21h