Travel Medicine and Infectious Disease 57 (2024) 102684

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Travel Medicine and Infectious Disease

journal homepage: www.elsevier.com/locate/tmaid

Artemisinin resistance-associated gene mutations in Plasmodium falciparum:

A case study of severe malaria from Mozambique
Daniela Casanova a, Vitória Baptista b, c, d, Magda Costa a, Bruno Freitas b, c,
Maria das Neves Imaculada Pereira b, c, Carla Calçada b, c, Paula Mota e, Olena Kythrich e, Maria
Helena Jacinto Sarmento Pereira a, Nuno S. Osório b, c, 1, Maria Isabel Veiga b, c, f, 1, *
a
Internal Medicine Department, Hospital Senhora da Oliveira, 4835-044, Guimarães, Portugal
b
Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal
c
ICVS/3B’s─PT Government Associate Laboratory, 4806-909, Guimarães/ Braga, Portugal
d
Microelectromechanical Systems Research Unit (CMEMS-UMinho), School of Engineering, University of Minho, Campus de Azurém, 4800-058, Guimarães, Portugal
e
Clinical Pathology Department, Hospital Senhora da Oliveira, 4835-044, Guimarães, Portugal
f
Clinical Academic Center-Braga (2CA-Braga), 4710-243, Braga, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: Background: The effectiveness of artemisinin-based combination therapies (ACT) in treating Plasmodium falcip­
Imported malaria arum, is vital for global malaria control efforts, particularly in sub-Saharan Africa. The examination of imported
Molecular markers cases from endemic areas holds implications for malaria chemotherapy on a global scale.
Resistance
Method: A 45-year-old male presented with high fever, dry cough, diarrhoea and generalized muscle pain,
Artemisinin-based combination therapy
following a two-week trip to Mozambique. P. falciparum infection with hiperparasitemia was confirmed and the
Mozambique
patient was treated initially with quinine and doxycycline, then intravenous artesunate. To assess drug sus­
ceptibility, ex vivo half-maximal inhibitory concentration assays were conducted, and the isolated P. falciparum
genome was deep sequenced.
Results: The clinical isolate exhibited elevated ex vivo half-maximal inhibitory concentration values to dihy­
droartemisinin, lumefantrine, mefloquine and piperaquine. Genomic analysis identified a I416V mutation in the
P. falciparum Kelch13 (PF3D7_1343700) gene, and several mutations at the Kelch13 interaction candidate genes,
pfkics (PF3D7_0813000, PF3D7_1138700, PF3D7_1246300), including the ubiquitin carboxyl-terminal hydrolase 1,
pfubp1 (PF3D7_0104300). Mutations at the drug transporters and genes linked to next-generation antimalarial
drug resistance were also present.
Conclusions: This case highlights the emergence of P. falciparum strains carrying mutations in artemisinin
resistance-associated genes in Mozambique, couple with a reduction in ex vivo susceptibility to ACT drugs.
Continuous surveillance of mutations linked to drug resistance and regular monitoring of drug susceptibility are
imperative to anticipate the spread of potential resistant strains emerging in Mozambique and to maintain
effective malaria control strategies.

1. Introduction endorsement by the World Health Organization (WHO) in 2001. The

Malaria, caused by Plasmodium falciparum, remains a significant
public health burden worldwide, with 247 million cases reported in
2021 with sub-Saharan Africa bearing the greatest burden, accounting
for about 95 % of all malaria cases [1]. Artemisinin-based combination
therapies (ACT) have been the mainstay of malaria treatment since their
unique combination of artemisinin derivatives’ rapid action and the
longer-lasting effect of partner drugs has contributed to a substantial
reduction in malaria-related morbidity and mortality over the past two
decades. However, the emergence of P. falciparum strains with reduced
susceptibility to ACT drugs poses significant threats with heavy impact
on the cost of global malaria control. Furthermore, it escalates the

* Corresponding author. Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057, Braga,
Portugal.
E-mail address: mariaveiga@med.uminho.pt (M.I. Veiga).
1
Shared last authorship.

https://doi.org/10.1016/j.tmaid.2023.102684
Received 25 October 2023; Received in revised form 6 December 2023; Accepted 26 December 2023
Available online 29 December 2023
1477-8939/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
D. Casanova et al. Travel Medicine and Infectious Disease 57 (2024) 102684

demand for the development of new drugs to replace artemisinin in the (Glasgow Coma Scale 15) with no focal neurological deficits, and both
event of their global failure. In addition, patients whose treatment fails Kernig and Brudzinky signs were negative, ruling out meningitis. His
due to infection with resistant strains, beside increasing the probability vital signs revealed afebrile status (37.4 ◦ C), hypotension (83/46
of developing severe symptoms, require repeated consultations at health mmHg), and an elevated heart rate (134 bpm). Physical examination
facilities, increasing health care costs and resulting in more days of daily revealed pale and dehydrated mucosa along with generalized jaundice.
activities absenteeism (such as employment and school). On pulmonary auscultation, pulmonary sounds were presented bilater­
Artemisinin resistance has been extensively documented in South­ ally, and no extra sounds were noted. On abdominal exam, augmented
east Asia, resulting in diminished efficacy of ACT and an increased risk hydro-aerial noises were present, but no pain at palpation or peritoneum
of treatment failure [2,3]. Clinical resistance to artemisinin has been irritative signs were noted. Blood analysis showed direct hyper­
defined as increased parasite clearance time [2] and is correlated with bilirubinemia, severe thrombocytopenia, hepatic cytolysis, rhabdo­
decreased in vitro parasite susceptibility, expressed in the ring-stage myolysis, coagulopathy with elevated lactate dehydrogenase, increased
survival assay (RSA) [4]. This clinical and biological phenotype is pri­ levels of reactive C protein, and acute kidney injury (Table 1). Arterial
marily associated with single nucleotide polymorphisms (SNP) affecting blood gas analysis revealed hyperlactatemia with mild acidosis, while
the parasite kelch 13 propeller protein; PfK13 [5,6]. While the exact no hypoglycemia was observed.
function of the PfK13 remains to be determined, it was found to Blood, urine and stools cultures were performed to rule out enteric
contribute to the endocytic transport of hemoglobin to the parasite sepsis. It was also made a panel of respiratory virus, namely influenza,
digestive vacuole [7]. PfK13 SNPs associated with drug resistance were Respiratory Syncytial Virus (RSV) and SARS-CoV2, all being negative.
shown to reduce protein concentration [7] and decreased pfk13 tran­ Microscopic examination of a Giemsa-stained thin blood smear
scription reported to lead to longer in vivo clearance upon indicated the presence of P. falciparum-infected red blood cells, with a
artemether-lumefantrine treatment [8]. Reduced PfK13 in turn leads to parasitemia level of 30 % (Fig. 1).
a reduced hemoglobin uptake, lowering the accumulation of toxic haem, The patient was diagnosed with severe P. falciparum malaria ac­
the activator of artemisinin, and consequently supports therapy resis­ cording to the criteria set by the WHO [10]: hyperbilirubinemia with
tance through reduced activation. The recent reports of the emergence jaundice, metabolic acidosis, coagulopathy with elevated lactate dehy­
of artemisinin partial resistance in Africa [9] with the apparent rapid drogenase, and a parasitemia of 30 %. He was admitted to a level 2 care
spread of mutations at the pfk13 gene associated with artemisinin partial unit in the hospital. Due to the unavailability of artesunate, the patient
resistance is of great concern. Worryingly, this emergence has not spread initiated antimalarial treatment with 1400 mg of quinine gluconate as
from Southeast Asia but has appeared de novo. the loading dose administered intravenously followed by 850 mg 12 h
Mozambique, a southeastern African country represents a high after and doxycycline 100 mg twice daily on the first day. On the second
burden high impact country defined by WHO, accounting alone for 4 % day, the patient’s treatment was switched to artesunate 2.4 mg/kg
of all malaria cases worldwide. Due to the threat of artemisinin resis­ administered 12/12 h intravenously on the first day and quinine glu­
tance, monitor the performance of ACT drugs and molecular surveil­ conate and doxycycline were discontinued, followed by artesunate 2,4
lance of the P. falciparum genome as markers of resistance to
artemisinins and the ACT partner drugs becomes of crucial importance
in this region. Table 1
In this study, we present a case of severe P. falciparum infection Blood analysis results.
imported by a traveler returning from Mozambique, who exhibited ex Days post hospital admission
vivo reduced susceptibility to most ACT drugs, along with mutations at EVALUATED 1st 2nd 3rd 4th 5th 23rd
the artemisinin resistance linked genes. This case emphasizes the urgent PARAMETER day day day day day day
need for continuous surveillance of antimalarial drug susceptibility and
HEMOGLOBIN (G/DL) 15.3 9.7 10.0 10.9 8.7 8.5
the timely adaptation of treatment guidelines to preserve the efficacy of LEUKOCYTES (X103/ 9.0 8.3 8.9 12.9 18.9 10.5
current malaria control strategies. By closely monitoring drug resistance UL)
patterns and promptly updating treatment protocols, we can effectively NEUTROPHILS (X103/ 7.7 6.2 5.5 7.6 13.0 7.9
UL) 0.9 1.8 2.4 4.3 4.1 1.7
mitigate the spread of resistant strains and maintain the effectiveness of
LYMPHOCYTES
ACTs in the fight against malaria. (X103/UL)
PLATELETS (X103/UL) 12 32 36 63 47 253
2. Material and methods CREATININE (MG/DL) 2.67 3.31 4.86 5.71 2.51 1.71
SODIUM (MEQ/L) 135 136 137 138 133 144
POTASSIUM (MEQ/L) 3.16 3.98 4.24 3.94 3.22 3.27
2.1. Case presentation TOTAL BILIRUBIN/ 7.44/ 7.03/ 13.38/ 12.14/ 8.77/ 1.65/
DIRECT BILIRUBIN 5.52 5.54 10.07 9.88 6.8 1.22
In 2021, a 45-year-old male presented to the emergency department (MG/DL)
at Hospital Senhora da Oliveira in Guimarães, Portugal, with three days TGO/TGP 143/ 209/ 462/ 447/ 172/ 39/
(GLUTAMIC- 130 292 301 320 216 104
fever as his main complaint. He reported returning from a one-month
OXALACETIC
stay in Mozambique five days before being admitted to the hospital. TRANSAMINASE/
No information regarding the patient’s residence area in Mozambique GLUTAMIC
was collected. In addition to a persistent high fever occurring every 4 h, PYRUVIC
unresponsive to antipyretics (paracetamol 1000 mg three times a day TRANSAMINASE)
(U/L)
and ibuprophen 600 mg two times a day), the patient also experienced a LDH (LACTATE 746 992 2478 2058 1117 297
dry and irritative cough that remained constant over time, without DEHYDROGENASE)
dyspnoea, pleuritic chest pain, nasal congestion, or a sore throat. (U/L)
Furthermore, the patient reported diarrhoea with liquid stools approx­ PCR (C-REACTIVE 107.1 143.8 195.4 311 260 8.1
PROTEIN) (MG/L)
imately four times a day, without any presence of blood, mucus,
MIOGLOBIN (NG/ML) 2087 451 346 360 281 –
abdominal pain, or alterations in gastrointestinal transit. Additionally, PT/APTT (PARTIAL 16.5/ 15.3/ 13.4/ 12.7/ 13/ 15.3/
the patient mentioned generalized muscle pain, all of which had been THROMBOPLASTIN 30.6 28.3 28.8 28.7 28.2 23.7
present for the same duration of three days. The patient confirmed that TIME) (SEC)
he had not taken any malaria chemoprophylaxis. BLOOD SMEAR 30 30 <1 <1 <1 0
PARASITEMIA (%)
Upon arrival at the emergency department, the patient was alert

2
D. Casanova et al.
Fig. 1. Timeline workflow of patient treatment and infection level post hospital admission. Microscopy images were obtained using an Olympus BX61 microscope
coupled with a digital camera (DP70) and the Olympus cellSens Dimension 1.18 software. QN: quinine; DOX: doxycycline; ATS: artesunate.
mg/kg once daily for 5 days (Fig. 1).
On the first day, the patient experienced observed seizures and
progressively became more somnolent and disoriented, with a Glasgow
Coma Scale score of 7, despite a normal cranio-encephalic computed
tomography. A normal blood sugar reading within the range of 120–172
mg/dL has excluded the presence of neuroglycopenia. Due to cerebral
malaria and significant neurological depression, the patient was trans­
ferred to the intensive care unit and required invasive ventilation.
Concurrently, there was a decline in laboratory parameters, with a peak
lactate dehydrogenase level of 2478 IU/L and a total bilirubin peak of
13.38 mg/dL on day 3, including renal dysfunction necessitating dial­
ysis, with peak creatinine levels reaching 5.71 mg/dL on day 4. The
patient also developed anemia with 6,9 g/dL hemoglobin on day 6 and
received two units of red blood cell concentrate. The parasitemia
resolved by day 3 following the initiation of treatment. With improved
neurological status, the patient was extubated on day 13, exhibiting no
neurological sequelae. He was discharged on day 23, with all dysfunc­
tions resolved, and referred to our hospital for follow-up.
2.2. Collection of P. falciparum clinical isolate and ethics
Venous blood was collected to BD Vacutainer EDTA Blood Collection
Tube before the treatment started. The blood sample was kept at 4 ◦ C
until transported to University of Minho within 24 h of blood draw.
Approval for the study was obtained by the health ethic commission of
Hospital Senhora da Oliveira in Guimarães (ref. 83/2019) and by the
Life and Health Science Research ethic commission of University of
Minho (CEICVS 24/2020).
2.3. P. falciparum clinical isolate ex vivo drug susceptibility assay
The whole blood sample was initially transferred into a micro­
centrifuge tube. Following this, the sample underwent centrifugation at
7000×g for 2 min, and the plasma and buffy coat were carefully aspi­
rated and removed. The red blood cells (RBC) were subsequently washed
twice with malaria culture medium (consisting of RPMI 1640 medium
supplemented with 2 mM l-glutamine, 200 μM hypoxanthine, 0.25 μg/
ml gentamicin, 25 mM HEPES, 0.2 % NaHCO3, and 0.25 % Albumax II
from Invitrogen; ThermoFisher Scientific). Parasitemia was adjusted to a
range between 0.2 and 0.4 % and hematocrit was adjusted to 1 % using
healthy human O + RBC (Portuguese Institute of Blood & Transplant,
IPST). The culture mixture was then distributed across three technical
3
Travel Medicine and Infectious Disease 57 (2024) 102684
replicates of pre-dosed 96-well drug plates and incubated for 72 h under
a specific gas composition (92 % N2, 5 % CO2, 3 % O2) at 37◦ C within a
modular incubator chamber (Billups-Rothenburg Inc.). The drug sus­
ceptibility assay of the clinical isolate was run in parallel with the
P. falciparum 3D7 strain (MRA-102; BEI-resources), synchronized at the
ring stage, and prepared with the same parasitemia and hematocrit
levels. Both were distributed across three technical replicates of pre-
dosed 96-well drug plates and incubated for 72 h under the same
modular incubator chamber. Drug plates were prepared with titrated
concentrations of dihydroartemisinin (DHA) (SIGMA; D7439), lume­
fantrine (LMF) (SIGMA; L5420), monodesethyl-amodiaquine (md-AQ)
(SIGMA; SMB00947), mefloquine (MFQ) (SIGMA; M2319), piperaquine
(PPQ) (SIGMA; C7874), pyronaridine (PND) (SIGMA; P0049) and the
drugs cipargamin (MCE; HY-14430), artefenomel (MCE; HY-16762),
ferroquine (MCE; HY-135847) and DSM265 (MCE; HY-100184) pres­
ently at the product development stage [11].
Growth was measured based on measuring SYBR®Green I fluores­
cence on optimized WWARN INV08 assay [12] after freeze-thaw using
4x SYBR Green I on a fluorometer after 3 h of incubation [13]. IC50
values were calculated by using non-linear regression curve-fitting al­
gorithm log(inhibitor) vs. normalized response–variable on GraphPad
Prism 7 software. Statistical significance was assessed by comparing the
IC50 antimalarial responses of the P. falciparum clinical isolate (HSOG3)
with the 3D7 reference strain. The analysis was performed on a single
biological replicate, with technical triplicates, using a parametric
two-tailed, Welch t-test. The Benjamin, Krieger and Yekutieli method
was employed for multiple testing correction. The statistical analysis
was conducted using GraphPad Prism 7 software and significance was
recognized for p ≤ 0.05.
2.4. Genomic analysis
DNA was extracted from venous blood sample using the NZY Tissue
gDNA Isolation kit (Nzytech, Lda) according to the manufacturer’s in­
structions. To improve the concentration of parasite DNA and deplete
most of the human DNA, prior to DNA extraction, the fresh sample was
centrifuged to remove plasma and most of the white blood cells. The
extracted DNA was sent to BGI company for whole genome sequencing
using PE100 DNBseqTM sequencing.
After sequencing, bioinformatic analysis was performed using the
Genome Analysis Toolkit (GATK). We used a variant calling pipeline
from gencorefacility/variant-calling-pipeline-gatk4, implemented in
D. Casanova et al.
Nextflow (version 21.10.6) and run with Docker. The pipeline used
HaplotypeCaller version 4.1.9.0. and other tools for quality control,
alignment, duplicate marking, base recalibration, variant calling, and
filtering steps. The quality control filters included FS_filter, MQRank­
Sum_filter, MQ_filter, QD_filter, ReadPosRankSum_filter, and SOR_filter.
We annotated the variants using SnpEff (version 4.3). We analyzed SNPs
related to drug resistance and phylogeography using custom Python
code and scikit-allel. We manually curated a list of drug resistance genes
from the literature and used a list of phylogeographic informative SNPs
from malaria-db GitHub repository. We compared our isolate with a
global dataset of P. falciparum samples from the MalariaGEN Community
Project (Pf7 dataset), which contains genotype calls on 10 million SNPs
and indels across 20,864 samples [14]. We selected a subset of 10,348
samples with an Fws value of ≥0.95 and calculated the allele frequencies
for each population using custom Python code and xarray. The data and
code used in the bioinformatics analyses were deposited on GitHub
(https://github.com/nunososorio/Pfalciparum-ReducedACT-Susceptibi
lity).
Gene copy number variation of pfpm2 (coding for plasmepsin 2),
pfpm3-1 (coding for the hybrid plasmepsin 3-1) and pfmdr1 (coding for
multidrug resistance 1) was performed with ΔΔCt methods as described
in Refs. [13,15], respectively, using P. falciparum 3D7 strain as the
reference of single gene copy. The gene edited lines Dd2PM2 and Dd2PM3
published elsewhere [13] were used as controls of gene duplication for
PM2 and PM3, respectively, and Dd2 wild type as with 3–4 pfmdr1 gene
copy number.
Fig. 2. Inhibition concentration curves of antimalarial dihydroartemisinin, lumefantrine, mefloquine, monodesethyl-amodiaquine, piperaquine and pyronaridine
determined for P. falciparum strain 3D7 (shown in black line) and the clinical isolate HSOG3 (shown in blue); graphs depict n = 3 technical point each; error bars
indicate ± standard error of mean (SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4
Travel Medicine and Infectious Disease 57 (2024) 102684
3. Results
3.1. Ex vivo antimalarial drug response
The clinical isolate (HSOG3) from Mozambique was successful
evaluated for ex vivo half-maximal inhibitory concentration drug
response to all drugs tested (Figs. 2 and 3). The assay was done in par­
allel with the P. falciparum 3D7 reference strain synchronized at ring
stage. The arithmetic mean values of the HSOG3 IC50s estimates
(±SEM, standard error of the mean) for DHA was 2.30 nM (±0.032),
LMF was 11.72 nM (±0.783), MFQ was 79.16 nM (±5.395), md-AQ was
14.23 nM (±0.369), PPQ was 11.68 nM (±0.537) and PND was 2.43 nM
(±0.155). For drugs that are presently under development the IC50 es­
timates presented for DSM265 was 4.66 nM (±1.184), Artefenomel was
4.77 nM (±0.394), Cipargamin was 0.65 nM (±0.012) and Ferroquine
was 4.56 nM (±0.344). Comparing the IC50s of the isolate with the
P. falciparum 3D7 strain, differences in susceptibility were detected
(Table 2). To highlight, the HSOG3 presented a 2-fold decrease sus­
ceptibility to DHA, more than 5-fold decreased susceptibility to LMF and
MFQ and 4-fold decreased susceptibility to PPQ when compared with
the P. falciparum 3D7 strain IC50 values. In contrast, the HSOG3 when
compared with 3D7, presented similar susceptibility to md-AQ and to
the new antimalarial compounds, DSM265, artefenomel, cipargamin
and ferroquine, presently at the product development stage (Fig. 3 and
Table 2).
3.2. Genome sequencing of HSOG3
To elucidate the genetic variations associated with parasite drug
response and to ascertain the presence of SNPs that discriminate be­
tween P. falciparum phylogeographic lineages, we conducted whole-
D. Casanova et al. Travel Medicine and Infectious Disease 57 (2024) 102684

Fig. 3. Inhibition concentration curves of antimalarial product under development stage, DSM265, artefenomel, ferroquine and cipargamin determined for
P. falciparum strain 3D7 (shown in black line) and the clinical isolate HSOG3 (shown in blue); graphs depict n = 3 technical point each; error bars indicate ± standard
error of mean (SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

observed a duplication rate of approximately 19.39 %. The mean insert

Table 2
size was calculated to be 234.13. In terms of variant discovery, we
IC50 values (mean ± SEM) obtained for P. falciparum clinical isolate and 3D7
identified 79,252 SNPs prior to filtering and base quality score recali­
reference strain, for dihydroartemisinin (DHA), lumefantrine (LMF), mefloquine
(MFQ), monodesethyl-amodiaquine (md-AMQ), piperaquine (PPQ), pyronar­ bration (BQSR). Following BQSR and subsequent filtering, this number
idine (PND), and for drugs under development. was reduced to 37,956 SNPs. Most SNPs that did not pass QC filters were
located at the extremities of the chromosomes (Figure A1). These re­
IC50 mean nM (±SEM) P-valuea
gions are known for their high variability which can lead to challenges in
HSOG3 3D7 accurately mapping and aligning sequencing reads. This could poten­
DHA 2.30 (±0.032) 1.14 (±0.033) 0.0000152 tially result in a higher rate of SNPs failing quality control filters. The
LMF 11.72 (±0.783) 1.34 (±0.413) 0.001 average coverage depth across the genome in our dataset was found to
MFQ 79.16 (±5.395) 13.49 (±6.565) 0.002
be approximately 2652.72x. Overall, the generated sequencing data
md-AMQ 14.23 (±0.369) 16.31 (±3.056) 0.567
PPQ 11.68 (±0.537) 2.56 (±0.797) 0.001 provides valuable insights into the genetic variations of the HSOG3
PND 2.43 (±0.155) 1.46 (±0.023) 0.023 clinical isolate.
DSM265 4.66 (±1.184) 3.44 (±0.203) 0.412
Artefenomel 4.77 (±0.394) 4.77 (±0.105) 0.994
Cipargamin 0.65 (±0.012) 0.50 (±0.041) 0.060 3.3. SNP-based analysis of phylogeographic lineage
Ferroquine 4.56 (±0.344) 4.25 (±0.139) 0.471
a
Statistical significance (multiple t-test) determined using the Welch t-test Determining the phylogeographic lineage of P. falciparum is chal­
and Benjamin, Krieger and Yekutieli method, with alpha = 0.05. Each row was lenging. Despite being susceptible to bias, particularly in moderate-to-
analyzed individually, without assuming a consistent SD. high transmission areas in Africa where P. falciparum exhibits the
greatest genomic diversity, the strategy most commonly employed relies
genome sequencing of the HSOG3 clinical isolate. The DNA for this on the use of a select set of SNPs identified as discriminative between
analysis was extracted from a venous blood sample, following the lineages [16]. In this study, we utilized the
removal of plasma and most white blood cells. The extracted DNA was phylogeographically-informative list of 50 SNPs from malaria-profiler
sequenced using PE100 DNBseqTM technology. (https://github.com/jodyphelan/malaria-db) to characterize HSOG3.
This process yielded a total of 609,426,842 reads. Of these, To evaluate the reliability of these SNPs, we cross-referenced them with
546,964,368 reads (approximately 89.75 %) were successfully aligned, the “Pf7 dataset” from the MalariaGEN P. falciparum Community Project
accounting for 72,482,145,039 bases with an average read length of (version 7, https://www.malariagen.net/apps/pf7/). We examined
150. Notably, most of the reads (99.76 %) were paired. However, we whether the presence of these SNPs corresponded with the annotated
phylogeographic lineage. From the 16,203 whole-genome sequencing

5
D. Casanova et al. Travel Medicine and Infectious Disease 57 (2024) 102684

samples in the “Pf7 dataset” that passed quality control, we selected 10,
348 samples with an Fws value of ≥0.95 for this analysis. The Fws metric
was employed to select samples likely to contain a single genotype,
thereby minimizing potential confusion arising from a high multiplicity
of infection. According to previous studies ([17–21], an infection is
considered to predominantly contain a single genotype when Fws
≥0.95.
Our analysis of the sequencing data revealed that HSOG3 contains
five SNPs from the list of 50 phylogeographically informative SNPs
tested, all of which are attributed to P. falciparum isolates from Eastern
Africa [22]. These SNPs were sequenced with high depth and quality
scores, as detailed in Table 3. Upon cross-referencing these SNPs with
their population frequency in the Pf7 dataset, we further supported that
the combined presence of these SNPs strongly suggests that HSOG3 is an
isolate of African origin (Table 3). More specifically, it is highly unlikely
that HSOG3 originates from Western Africa, but rather, it appears to be
derived from the Eastern African lineage (Table 3).
The identification of the Eastern African lineage in this sample is
consistent with the presumed geographic origin of the infection. The
patient had recently traveled to Mozambique, which is known to be an
endemic region for the Eastern African lineage of the parasite. Similarly
to HSOG3, the isolates in Pf7 dataset from Mozambique are all classified
as East-African. Thus, our results are in line with the expected lineage
based on the patient’s travel history report.

3.4. Analysis of genetic basis of antimalarial drug resistance

Table 3
Phylogenetically informative SNPs present in HSOG3.
Disclosing the genetic variations associated with parasite drug
Chromosome Pf7 dataset (n = HSOG3 response is key for the improvement of molecular surveillance of drug
10,348)
resistant parasites. We selected a list of 37 genes containing relevant
Position Frequency of the Allele: Depth Quality drug resistance information including the artemisinin-resistance pre­
alternative allele (reference allele/ Score disposing background [23] and newly revealed genes involved in the
by lineagea (%) alternative allele)
PfK13- endocytosis pathway as potential alternative for artemisinin
01 489337 AF-C 87.18 A: 0/C: 1259 31596 resistance candidates [7]. Beside the selected genes, we searched for the
AF-NE 81.65
hybrid sequence created because of the plasmepsin 2/3 duplication
AF-E 81.03
OC-NG 58.51 associated with PPQ decreased susceptibility [24].
SA 56.85 From the selected genes listed in Table 4 with relevant context in
AS-S-E 6.15 drug resistance, 20 out of 37 genes presented no non-synonymous
AF-W 2.79
mutations.
AS-S-FE 2.13
AS-SE-E 1.11
The HSOG3 clinical sample presented several potential artemisinin
AS-SE-W 0.32 resistance-linked gene polymorphisms. At the pfk13 gene, a I416V SNP
02 375427 AF-E 82.21 G: 3/A: 4356 131207 was detected. This SNP is localized in the highly conserved BTB/POZ
AF-C 68.24 domain of the PfK13 protein [25]. Several polymorphisms were also
AF-NE 48.62
found at the linked K13 compartment [7]. This included polymorphisms
OC-NG 22.06
AS-SE-E 12.06 at the pfubp1, the pfap2-mu, the pfcoronin, the pfmca2 and at the newly
AS-SE-W 8.62 identified PfK13 interaction candidates (KICs), the pfkic4, pfkic5 and
AS-S-FE 3.58 pfkic7.
AF-W 3.45 Non-synonymous mutations at the PfCRT and PfMDR1 drug trans­
AS-S-E 3.08
SA 0
porters, well-known modulators of multidrug resistance, were not
03 383169 AF-E 74.75 A: 1/T: 2506 74112 detected, with the synonymous mutations Asn504Asn (aat-aac) and
AF-C 71.24 Thr1069Thr (act-acg) present in PfMDR1. At the sodium/hydrogen
AF-NE 31.19 exchanger transporter a SNP inside the ms4760 microsatellite (K1380 N)
OC-NG 13.52
was found, associated with quinine resistance. The clinical isolate also
AS-S-FE 7.38
AS-S-E 3.85 presented 4 SNPs at the PfMRP2 and 3 SNPs at the PfABCI3, known to
AF-W 2.86 mediate resistance to experimental antimalarial compounds [26–28].
SA 2.07 Polymorphisms associated with PPQ resistance such as at the codon
AS-SE-W 0.77 415 of pfexo and the plasmepsin2/3 breakpoint [29], were not detected
AS-SE-E 0.61
09 596674 AF-E 83.66 T: 0/C: 3891 103670
in the HSOG3 P. falciparum isolate. Gene copy number variation of PM2
AF-C 72.53 and PM3, analyzed through realtimePCR ΔΔCT method, revealed single
SA 58.62 gene copy for both pfpm2 and pfpm3-1 as well as for pfmdr1 (Fig. 4).
AF-NE 46.79 Polymorphims at the PfDHFR and PfDHPS, associated with sulpha­
AS-S-E 20.61
doxine – pyrimethamine resistance were detected in the HSOG3
OC-NG 11.35
AS-S-FE 8.83 P. falciparum isolate, presenting the triple mutant haplotype for PfDHFR
AF-W 2.92 (S108 N/N51I/C59R) and the SNP S436A and G437A at the PfDHPS.
AS-SE-E 0.71 At other relevant drug resistance linked genes, the HSOG3 presented
AS-SE-W 0.38 a non-synonymous mutation at the PfCARL (Lys903Glu) and 3 out of 8
13 2481108 AS-SE-W 92.46 C: 0/G: 4673 124963
AS-SE-E 89.86
mutations detected at the PfPI4KB were non-synonymous (His24Leu,
AS-S-FE 88.26 Val100Ala, Gly124Asp, Asp875Asn).
AF-E 85.24
AF-C 81.62 4. Discussion
OC-NG 78.29
AF-NE 63.30
AS-S-E 36.92 This study presents a case of severe P. falciparum malaria imported
SA 24.14 from Mozambique, characterized by mutations in artemisinin
AF-W 4.84 resistance-associated genes. Additionally, there is observed ex vivo sus­
a
AF-C: Africa - Central; AF-E: Africa - East; AF-NE: Africa - Northeast; AF-W: ceptibility to several drugs commonly used in ACT, as compared to the
Africa - West; AS-SE-E: Southeast Asia - East; AS-SE-W: Southeast Asia - West; 3D7 strain.
AS-S-E: South Asia - East; AS-S-FE: South Asia - Far East; OC-NG: Oceania - New The movement of malaria in endemic countries has contributed to
Guinea; SA: South America. the spread of drug resistance and threatens long-term eradication goals.

6
D. Casanova et al. Travel Medicine and Infectious Disease 57 (2024) 102684

Table 4 Table 4 (continued )

Drug resistance genes and genetic polymorphisms found. GENE ID GENE EXTENDED GENE NAME NON-
GENE ID GENE EXTENDED GENE NAME NON- NAME SYNONIMOUS
NAME SYNONIMOUS MUTATIONa
MUTATIONa
PF3D7_0417200 DHFR- bifunctional dihydrofolate Asn51Ile
ARTEMISININ RESISTANCE LINKED GENES TS reductase-thymidylate
PF3D7_1343700 K13 kelch protein K13 Ile416Val synthase
PF3D7_0104300 UBP1 ubiquitin carboxyl-terminal Asp287Asn Cys59Arg
hydrolase 1 Ser108Asn
Tyr615His PF3D7_0810800 PPPK- hydroxymethyldihydropterin Ser436Ala
Arg1133Ser DHPS pyrophosphokinase-
Glu1531Asp dihydropteroate synthase
Lys1914Asn Gly437Ala
Glu1915Lys PIPERAQUINE RESISTANCE LINKED GENES
Arg2238Lys PF3D7_1362500 EXO 3′-5′ exonuclease None
PF3D7_0813000 KIC7 Kelch13 interaction candidate Asn434Ser PF3D7_1408000 PMII plasmepsin II Asp231Asn
KIC7 Gln442His
PF3D7_1025000 EPS15 Eps15-like protein None PF3D7_1408100 PMIII plasmepsin III None
PF3D7_1138700 KIC5 Kelch13 interaction candidate Asn496His OTHER DRUG RESISTANCE LINKED GENES
(PF11_0398) KIC5 PF3D7_0321900 CARL cyclic amine resistance locus Lys903Glu
Tyr965Asp protein
Tyr1218His PF3D7_0509800 PI4KB phosphatidylinositol 4-kinase His24Leu
Lys1720Glu beta
PF3D7_1204400 G37 sexual stage-specific protein None Val100Ala
G37 Gly124Asp
PF3D7_1218300 AP2- AP-2 complex subunit mu Val103Leu Asp875Asn
(PFL0885w) MU PF3D7_0109800 cPheRS phenylalanyl-tRNA synthetase None
PF3D7_1246300 KIC4 clathrin adaptor domain- Asn352His alpha subunit
(PFL2220w) containing protein PF3D7_0603300 DHODH dihydroorotate dehydrogenase None
PF3D7_1251200 coronin coronin Ile217Arg PF3D7_1213800 PRS prolyl-tRNA synthetase None
(PFL2460w) PF3D7_MIT02300 CYTB cytochrome b None
PF3D7_1318100 FD ferredoxin None a
Within the variability found, all with exception of PfK13 Ile416Val SNP are
PF3D7_1320600 RAB11a ras-related protein Rab-11A None
PF3D7_1438400 MCA2 metacaspase-2 Tyr179Asn presently annotated at the integrative database of the P. falciparum genome;
(PF14_0363) PlasmoDB (https://plasmodb.org/). WT: Wild type (reference strain 3D7).
Ser185Gly
Asn1104Asp
Immunity may play an important role in the emergence and trans­
Asn1135His
Tyr1358Ser
mission potential of resistant parasites [30]. Considering the imported
Ser1363Thr cases, the weakened host malaria immunity of the travelers could favor
Phe1807Leu the selection of parasite mutations associated with drug resistance with
PF3D7_1451100 eEF2 elongation factor 2 None reduced fitness cost. The imported case herein in study, was a severe
PF3D7_1460900 ARPS10 ribosomal protein S10, None
case with parasitemia above 30 % and clear signs of cerebral malaria
apicoplast
DRUG TRANSPORTER GENES with significant neurological depression, that lead to admitting the pa­
PF3D7_0112200 MRP1 multidrug resistance- None tient in the intensive care unit with invasive ventilation. The case pre­
associated protein 1 sented has no indication of clinical resistance or treatment failure. The
PF3D7_0319700 ABCI3 ABC transporter I family Ser1461Cys patient received intravenous artesunate and resolved the parasitemia in
member 1
Lys1973Asn
less than 3 days, showing clearance rate responsive to artesunate. What
Asn2043Lys is noteworthy in this clinical case is that one day before receiving
PF3D7_0523000 MDR1 multidrug resistance protein 1 None artesunate, the patient had been treated with two doses of quinine along
PF3D7_0709000 CRT chloroquine resistance None with doxycycline. Despite the parasitemia remaining high on the second
transporter
day before the initial artesunate dose (Fig. 1), the swift schizonticidal
PF3D7_1036800 AT1 acetyl-CoA transporter 1 Phe548Ile
(PF10_0360) action of quinine (an aryl amino alcohol drug) and the apicoplast-related
Thr459Ser mode of action of doxycycline administered in the two doses could
PF3D7_1113300 UGP UDP-galactose transporter None potentially eliminate or, at the very least, mask the impact of
PF3D7_1211900 ATP4 non-SERCA-type Ca2+ None artemisinin-tolerant parasites. Considering the possible effects of the
-transporting P-ATPase
PF3D7_1229100 MRP2 multidrug resistance- Phe195Leu
three antimalarial drugs on successful parasite clearance, we believe it is
associated protein 2 not accurate to draw a direct comparison between the clinical outcome
Ser711Cys of this case and the correlation between in vivo patient clearance rate,
Asn1166Thr susceptibility to artemisinins and parasite genetics.
Asp1188Glu
The genomic data of this clinical isolate revealed several interesting
PF3D7_1303500 NHE sodium/hydrogen exchanger Val950Gly
Gln1210Lys aspects. The phylogeographic lineage analysis, although challenging
Lys1380Asn due to be susceptible to bias, particularly in moderate-to-high trans­
Phe1557Ser mission areas in Africa where P. falciparum exhibits the greatest genomic
PF3D7_1339900 ABCB5 ABC transporter B family None diversity, placed the P. falciparum isolate into the Eastern African line­
member 5
PF3D7_1352100 ABCB6 ABC transporter B family None
age, corroborating with the clinical report of the patient where the
member 6 parasite was isolated.
PF3D7_1447900 MDR2 multidrug resistance protein 2 None While drug resistance cutoffs are not well-established for most an­
SULPHADOXINE – PYRIMETHAMINE RESISTANCE LINKED GENES timalarials, the ex vivo drug susceptibility data from the clinical
P. falciparum isolate revealed a distinct response to various drugs
commonly used in ACT, as demonstrated by the standard 72 h IC50
assay. Specifically, for DHA, the IC50 values showed a twofold decrease

7
D. Casanova et al.
Fig. 4. Quantitative PCR data presenting the copy number variation of pfmdr1, pfpm2 and pfpm3-1 on HSOG3, 3D7 reference strain and controls. Data are presented
as mean ± SEM.
in susceptibility compared with the reference strain 3D7. However, it is
important to acknowledge that these results do not directly imply
reduced susceptibility to artemisinins. A notable limitation of this study
is the absence of an ex vivo RSA, the standard method for evaluating
artemisinin susceptibility associated with delayed parasite clearance in
vivo. While the RSA was designed to differentiate DHA-resistant para­
sites in vitro and correlate better with patient clearance half-life than
IC50 values, it still has limitations in accurately reflecting this clinical
parameter, as indicated by previous studies [31]. The significance of
IC50 values for DHA lies in their ability to detect a shift in susceptibility,
especially at the later parasite stages generally considered susceptible to
artemisinin. Despite caution regarding directly linking IC50 data to
reduced susceptibility to artemisinins, it is noteworthy that the observed
fold change positions HSOG3 among the exclusive 25 % of isolates in a
study involving 440 Ugandan isolates, demonstrating a more than
twofold decrease in IC50 susceptibility to DHA compared to the 3D7
reference strain [32].
At the drug resistance genes analyzed, PfK13 and other potential
artemisinin resistance linked gene polymorphisms were found. A non-
synonymous mutation was detected at the pfk13 gene at amino acid
position 416 with a change of isoleucine to a valine, localized in the
BTB/POZ domain, upstream of the propeller domain. This mutation has
previously been reported in an efficacy study of artemether-
lumefantrine in Tanzania [33]. In the Pf7 dataset, the PfK13 I416V
SNP, was detected in 11 samples (Table A1). However, the SNP did not
pass the applied QC Filter. Furthermore, only 2 of these samples passed
the quality control filters for mixed species or low coverage (PT0297-C
and SPT15622) and were included in the final analysis set (Table A2).
The PT0297-C sample was from Malawi and belonged to the
African-East population, while the SPT15622 sample was from Tanzania
and belonged to the African-East population. A recent population
genomic study from Mozambique reported no evidence of selection near
pfk13, nor did it identify artemisinin resistance-conferring mutations in
the pfk13 gene [34]. Importantly, based on the available data, the pfk13
I416V mutation was only detected with confidence in a few samples,
suggesting that it is either a new target of drug selection or a mutation
with a high fitness cost that limits its spread among immuno-competent
individuals in endemic regions. Indeed, drug resistance mutations often
have a fitness cost. In the context of imported cases, the low malaria
immunity of travelers could allow the survival of parasites with
emerging mutations that may alter drug response and pose a challenge
when combined with compensatory mutations. The origin and distri­
bution of this SNP and its contribution to artemisinin reduced suscep­
tibility remains unclear and warrants further investigation.
Additionally, validated PfK13 SNPs of reduced susceptibility to
artemisinins are present in the propeller domain of PfK13, which comes
after the BTB/POZ domain. Consequently, the majority of research
attention has been directed towards the propeller domain leading to a
scarcity of data from selection experiments or epidemiological
8
Travel Medicine and Infectious Disease 57 (2024) 102684
surveillance studies to speculate on the frequency of SNPs in the PfK13
BTB/POZ domain within individuals of African genetic backgrounds.
But although this mutation has been poorly studied, its presence could
potentially affect the efficacy of artemisinin-based treatments. In an in
vitro artemisinin resistance selection study, selected parasites exhibited
enhanced survival to DHA in the RSA [25]. This parasite contained a
PfK13 P413A mutation, localized in the BTB/POZ domain that was
further validated to confer in vitro artemisinin resistance through
CRISPR/Cas9 gene editing in Dd2 strain [25]. While the PfK13 muta­
tions are intriguing, it is noteworthy that researchers have documented
instances of artemisinin-resistant parasites without K13 mutations [31],
including in East Africa [9,35]. The clinical isolate studied here, origi­
nating from Mozambique, also revealed the presence of seven
non-synonymous mutations at the PfUBP1, previously annotated at the
integrative database; PlasmoDB. PfUBP1 mutations have been linked
with delayed clearance [36]; mutated in artesunate-resistant rodent
malaria parasites [37]; involved in endocytic transport of hemoglobin
and in close proximity with PfK13 [7]. Also involved in the endocytic
transport of hemoglobin and in proximity with PfK13 are the PfKIC4,
PfKIC5 and PfKIC7, the three proteins encompassing mutations in the
HSOG3 isolate. While the clinical significance of the mutations discov­
ered in genes linked to artemisinin resistance, such as the PfK13 I416V
SNP, remains uncertain, their detection underscores the importance of
continuous monitoring for mutations associated with drug resistance.
Drug transporter proteins are the hub of multidrug resistance
phenotype. Here we highlight the analysis of 12 transporter genes pre­
viously reported to play a role in resistance, including the canonical
PfMDR1 and PfCRT transporters located at the membrane of the diges­
tive vacuole with opposite flux. These transporters are responsible for
the collateral drug sensitivity in which, mutant isoforms of PfMDR1 and
PfCRT cause LMF and MFQ to remain in the cytosol of the parasite
instead of sequestering within the digestive vacuole [38]. Both trans­
porters presented in this clinical isolate as wild type allele and single
gene copy for pfmdr1. The HSOG3 clinical isolate presented an ex vivo
decreased susceptibility to LMF of about 9-fold and MFQ of 6-fold, when
compared with the reference 3D7 strain (Fig. 2, Table 2). It’s important
to note that field isolates, especially in ex vivo assays, may test less
sensitive than laboratory controls, warranting caution when extrapo­
lating results [35]. However, it is noteworthy that when compared with
a comprehensive study on East African isolates, HSOG3 ranks among the
exclusive 15 % and 5 % of isolates demonstrating a more than fivefold
decrease in susceptibility to LUM and MFQ, respectively, when
compared to the 3D7 reference strain [32]. This observed fold change
aligns with a previously reported risk factor, namely, the presence of an
asparagine at amino acid position 86 in the PfMDR1 and a lysine at
amino acid position 76 in PfCRT [39], a correlation supported by
gene-edited validation [15,40,41]. Mozambique introduced
artemether-lumefantrine as the official first-line treatment back in 2009
[42], pressuring the parasite to go under selection of the PfMDR1 N86
D. Casanova et al.
and PfCRT K76. In line with this, a recent report on a large cohort from
Mozambique with 2251 samples genotyped revealed a prevalence of 99
% for both PfMDR1 N86 and PfCRT K76. Although robust molecular
markers of LMF resistance are yet to be unveiled, the bottleneck selec­
tion observed points for the need to closer monitoring of the combina­
tion efficacy in the country.
Transporters at the cytoplasm membrane of the parasite such as the
PfMRP1 and PfMRP2, may also play a role in drug resistance. Non-
synonymous SNPs in PfMRP1, particular the I876V variant, have been
observed in recurrent infections following artemether-lumefantrine
treatment [43] and sulfadoxine-pyrimethamine treatment [44],
whereas several microindels and SNPs have been described in PfMRP2,
potentially affecting the parasite clearance time during ACT treatment
[45]. The HSOG3 isolate contained several SNPs in PfMRP2, including
two SNPs in microindel IV as previously described [45]. However, cor­
relation with resistance remains to be validated.
The combination sulfadoxine-pyrimethamine remains widely rec­
ommended for intermittent preventive treatment against P. falciparum in
pregnant women, and Mozambique is no exception [42]. Genomic
analysis of HSOG3 identified four mutations that have been confidently
linked to drug resistance. Three of these mutations - Asn51Ile, Cys59Arg,
and Ser108Asn – were found in the PF3D70417200 (DHFR-TS) gene,
known to be associated with pyrimethamine resistance. Additionally,
Ser436Ala and Gly437Ala mutations were found in the PF3D70810800
(DHPS) gene, which is linked to sulfadoxine resistance. The genetic
profile of this isolate aligns with the high prevalence of these mutations
reported in the country [46].
Piperaquine resistance has primarily been associated with the
duplication of the Pfpm2 and Pfpm3 genes, which encode plasmepsin 2
and 3, respectively. These aspartic proteases are located in the parasite’s
digestive vacuole and play a role in the host hemoglobin degradation.
These duplications have been reported in Africa [47]. Additionally, a
SNP leading to a Glu415Gly substitution in a putative exonuclease has
also been linked to reduced PPQ susceptibility [29]. While these markers
alone have not been shown to induce the resistance phenotype, they
appear to contribute to a genetic background that favors the emergence
of novel pfcrt polymorphisms, which do decrease susceptibility to PPQ
[48]. Furthermore, a decreased pfmdr1 gene copy number has been
associated with reduced PPQ susceptibility in isolates from Thailand
[49], aligning with epidemiological data indicating a decrease in pfmdr1
copy numbers and an increase in plasmepsin 2/3 duplications after the
implementation of dihydroartemisinin-piperaquine therapy in South­
east Asia [50]. Consistent with molecular epidemiological data in
Southeast Asia, a multigenic architecture for PPQ resistance has been
proposed [13], enhancing our understanding of how PPQ resistance
develops. The HSOG3 clinical isolate exhibited a four-fold decrease in ex
vivo susceptibility to PPQ when compared with the reference strain 3D7,
as determined by the standard P. falciparum IC50 assay. Interestingly, it
did not show a clear correlation with its genotype, as it had a single gene
copy of Pfpm2, Pfpm3 and Pfmdr1, and no SNPs were detected in PfEXO,
PfMDR1 or PfCRT – variants that have previously been reported to
modulate PPQ response. It is worth noting that the 72-h ex vivo assay
performed in this study may not be optimal for distinguishing recru­
descent and non-recrudescent isolates in patients treated with
dihydroartemisinin-piperaquine [51], and therefore, may not be ideal
for evaluating PPQ susceptibility. Nonetheless, our data suggests that,
under constant 72-h PPQ pressure, the parasite exhibited increased
resistance to PPQ at later trophozoite and schizont stage compared with
3D7 strain. This further underscore Africa’s susceptibility to PPQ resis­
tance, particularly in the context of pfmdr1 single-copy backgrounds
being predominant on the continent.
Polymorphism in other drug resistance-linked genes has been iden­
tified, especially those relevant to next generation antimalarial drugs.
The clinical isolate harbors non-synonymous mutations in PfCARL and
the PfAT1, which is associated with reduced susceptibility to imidazo­
lopiperazines like KAF156 and GNF179 [52,53]. Additionally, four
9
Travel Medicine and Infectious Disease 57 (2024) 102684
polymorphisms were detected in PI4KB, which is linked to Imidazo­
pyrazines and quinoxalines that bind to the ATP-binding site of PI(4)K
and alter the intracellular distribution of PI(4)P [54].
The transporter PfABCI3, known for conferring resistance to the new
generation of carboxamide-containing compounds [55], exhibited three
non-synonymous SNPs in HSOG3 isolate. This gene appears to be highly
polymorphic, consistent with recent findings in isolates from Uganda
[56]. However, despite the presence of several mutations in these novel
drug resistance genes, the ex vivo drugs response to the new generation
drugs did not indicate decreased susceptibility (Fig. 3).
The ACTs are presently the primary treatment for uncomplicated
malaria, and resistance to these drugs represents a significant challenge
in the global endeavor to control and eliminate malaria. This study
serves as an alert to the emergence of P. falciparum strains carrying
mutations in artemisinin resistance-associated genes in Mozambique,
potentially bearing relevance to the treatment outcomes of ACTs. The
genomic analysis of the parasitic isolate revealed mutations previously
linked to pyrimethamine and sulfadoxine resistance. Moreover, it un­
veiled potentially crucial new mutations in genes associated with arte­
misinin resistance, as well as mutations at drug transporter,
sites—known hubs for multidrug resistance.
This discovery emphasizes the urgent need for continuous surveil­
lance, particularly focusing on mutations found outside the propeller
domain of PfK13 and in the recently identified genes within the PfK13-
endocytosis pathway [7]. This surveillance aims to detect drug
resistance-associated mutations and maintain continuous monitoring of
drug susceptibility.
By closely monitoring drug resistance patterns and promptly
updating treatment protocols, we can effectively mitigate the spread of
resistant strains and ensure the continued effectiveness of artemisinin-
based combination therapies in our fight against malaria.
Fundings
This work has been funded by National funds, through the Founda­
tion for Science and Technology (FCT) - project UIDB/50026/2020 and
UIDP/50026/2020 and by the projects, NORTE-01-0145-FEDER-
000039 and NORTE-01-0145-FEDER- 085468, supported by Norte
Portugal Regional Operational Programme (NORTE 2020), under the
PORTUGAL 2020 Partnership Agreement, through the European
Regional Development Fund (ERDF). By 2CA Braga 2020 grant to MIV
and a Research Grant 2021 of the European Society of Clinical Micro­
biology and Infectious Diseases (ESCMID) to MIV. M.I.V. thanks FCT for
her contract funding provided through 2020.03113.CEECIND. V.B.
thanks FCT for the studentship SFRH/BD/145427/2019. The authors
declare having no conflicts of interest.
Data references
The data and code used in the bioinformatics analyses were depos­
ited on GitHub (https://github.com/nunososorio/Pfalciparum-Reduce
dACT-Susceptibility).
CRediT authorship contribution statement
Daniela Casanova: Conceptualization, Investigation, Resources,
Writing – review & editing. Vitória Baptista: Conceptualization, Data
curation, Formal analysis, Investigation, Writing – review & editing.
Magda Costa: Conceptualization, Investigation, Resources, Writing –
review & editing. Bruno Freitas: Data curation, Formal analysis,
Investigation, Writing – review & editing. Maria das Neves Imaculada
Pereira: Investigation, Writing – review & editing. Carla Calçada:
Formal analysis, Investigation. Paula Mota: Investigation, Resources.
Olena Kythrich: Investigation, Resources. Maria Helena Jacinto
Sarmento Pereira: Conceptualization, Investigation, Resources, Su­
pervision, Writing – review & editing. Nuno S. Osório:
D. Casanova et al. Travel Medicine and Infectious Disease 57 (2024) 102684

Conceptualization, Data curation, Formal analysis, Funding acquisition,
Investigation, Writing – review & editing. Maria Isabel Veiga:
Conceptualization, Investigation, Project administration, Supervision,
Visualization, Writing – original draft, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Transplantação (IPST), particularly to Centro de Sangue e da Trans­
plantação do Porto for their assistance in providing healthy blood
samples for our research.
We also thanks to the patient who participated in this study for their
informed consent and cooperation, without which this research would
not have been possible.
The following reagent was obtained through BEI Resources, NIAID,
NIH: Plasmodium falciparum, Strain 3D7, MRA-102, contributed by
Daniel J. Carucci and Strain Dd2, MRA-150, contributed by David
Walliker.

Acknowledgements

We would like to thank to Instituto Português do sangue e

Appendix
Table A1
Sample Information and Sequencing Depth for the novel PfK13 Ile416Val SNP

Chrom Position Alternative Allele SNP Filter pass Depth Sample Country Source

13 1725752 C TRUE 4168 HSOG3 Portugal (traveler) This study

13 1725752 C FALSE 8 PA0190-C Guinea Pf7 dataset
13 1725752 C FALSE 7 PA0264-C Guinea Pf7 dataset
13 1725752 C FALSE 14 PT0154-C Malawi Pf7 dataset
13 1725752 C FALSE 14 PT0154-Cx Malawi Pf7 dataset
13 1725752 C FALSE 29 PT0297-C Malawi Pf7 dataset
13 1725752 C FALSE 39 PT0297-Cx Malawi Pf7 dataset
13 1725752 C FALSE 37 QP0097-C Cameroon Pf7 dataset
13 1725752 C FALSE 11 RCN00746 Bangladesh Pf7 dataset
13 1725752 C FALSE 11 SPT15622 Tanzania Pf7 dataset
13 1725752 C FALSE 15 SPT35127 Gambia Pf7 dataset
13 1725752 C FALSE 19 SPT35129 Gambia Pf7 dataset

Table A2
Sample Information for the isolates potentially harboring the PfK13 Ile416Val SNP in “Pf7 dataset”

Sample Country Year ENA All samples same case Population QC pass Exclusion reason Sample type

PA0190-C Guinea 2011 ERR063527 PA0190-C AF-W False Mixed_species gDNA

PA0264-C Guinea 2011 ERR063605 PA0264-C AF-W False Mixed_species gDNA
PT0154-C Malawi 2011 ERR234517 PT0154-C,PT0154-Cx AF-E False Mixed_species gDNA
PT0154-Cx Malawi 2011 ERR484640 PT0154-C,PT0154-Cx AF-E False Mixed_species gDNA
PT0297-C Malawi 2011 ERR248962 PT0297-C,PT0297-Cx AF-E True Analysis_set gDNA
PT0297-Cx Malawi 2011 ERR484560 PT0297-C,PT0297-Cx AF-E False Lower_covered_duplicate gDNA
QP0097-C Cameroon 2013 ERR577843 QP0097-C AF-W False Mixed_species gDNA
RCN00746 Bangladesh 2015 ERR2542430 RCN00746 AS-S-FE False Mixed_species sWGA
SPT15622 Tanzania 2014 ERR2892029 SPT15622 AF-E True Analysis_set sWGA
SPT35127 Gambia 1990 ERR2892690 SPT35127 AF-W False Mixed_species sWGA
SPT35129 Gambia 1990 ERR2892798 SPT35129 AF-W False Mixed_species sWGA

10
D. Casanova et al. Travel Medicine and Infectious Disease 57 (2024) 102684

Fig. A1. Distribution of Single Nucleotide Polymorphisms (SNPs) across each chromosome of Plasmodium falciparum in the sequenced clinical isolate. The x-axis
denotes the genomic position, while the y-axis indicates the depth of sequencing. SNPs that passed the filter criteria are depicted in green, while those that did not
pass are shown in red.

11
D. Casanova et al. Travel Medicine and Infectious Disease 57 (2024) 102684

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