Background
Artemisinin (ART) and related compounds provide the main class of anti-malarial drugs, and ART resistance in
Plasmodium falciparum is one of the greatest threats to global efforts to control, eliminate and eradicate malaria. To forestall emergence and spread of ART resistance it was recommended that ART and its derivatives be used only in combination with a partner drug as an ART combination therapy (ACT), with over 400 million ACT treatments dispensed annually. It is conservatively estimated that 116,000 additional deaths would occur annually in the event of widespread ART resistance, with annual health costs of US$32 million and productivity losses exceeding US$385 million [
1]. The magnitude of the health and economic threat posed by ART resistance serves as an urgent call to action to develop strategies that circumvent its spread [
2]. Doing so requires understanding the underlying mechanisms of ART resistance.
Currently, ART resistance is prevalent across the Greater Mekong Sub-region (GMS) and centered on Cambodia, where it was first detected in 2007 [
3,
4]. Concern for the spread of ART resistance outside of Southeast Asia led to the Tracking Resistance to Artemisinin Collaboration (TRAC) study that assessed and tracked ART resistance across 15 sites both in Asia and Africa [
5]. Using parasite clearance as a measure of resistance, the TRAC project confirmed that ART-resistant
P. falciparum was established in Cambodia, Laos, Myanmar, Thailand, and Vietnam.
Artemisinin resistance is indicated by either delays in parasite clearance from patients, or by increased in vitro parasite survival under dihydroartemisinin (DHA) in ring-stage survival assay (RSA
0–3h) [
6]. Sequencing parasites selected under increasing ART pressure identified
pfkelch13 as a critical gene for conferring ART resistance [
7]. Multiple mutations have been identified in this gene, and several key single nucleotide polymorphisms (SNPs) have been validated using a gene-editing approach [
8,
9]. However, the function of
PfKelch13 and its role in ART resistance remain unclear.
Recent reports confirm that specific
pfkelch13 mutations confer ring-stage survival [
7,
8], and indicate possible mechanisms of ART resistance, including perturbation of haemoglobin processing [
10‐
12], protein ubiquitination [
13], increased expression of oxidative stress response [
13,
14], unfolded protein response pathways [
14], or phosphatidylinositide 3-kinase pathways [
15]. While these results may all be relevant, a full understanding of the mechanisms involved in ART action and resistance has yet to emerge [
16,
17].
One of the key partner drugs used with ART for malaria treatment in the GMS is piperaquine (PPQ), which has proved well tolerated and highly effective in areas where multi-drug-resistant
P. falciparum is prevalent. However, emergence of PPQ resistance threatens to undermine this strategy in areas of increasing ART resistance [
18,
19]. Currently PPQ resistance is evident by higher PPQ half maximal effective concentration (EC
50) values and elevated recrudescence rates in settings where DHA–PPQ is in use and ART resistance is common [
18,
19]. Reported molecular markers of PPQ resistance include SNPs in
pfcrt and de-amplification of a region on chromosome 5 that includes or is proximal to
pfmdr1 [
20]. PPQ resistance is also associated with an amplification of
plasmepsin II and
III [
21,
22]. Yet, to date no mechanism has been established for PPQ resistance. Increasing resistance to both ART and PPQ in the same parasite population has motivated a call for use of triple combination therapy in certain malaria-endemic settings where ACT may have reduced efficacy [
18,
23]. Specifically, mefloquine (MFQ) has been suggested for combination with ART and PPQ given the inverse effects PPQ and MFQ pressure have on
pfmdr1 copy number variation (CNV), as PPQ negatively selects
pfmdr1 CNV, while MFQ positively selects
pfmdr1 CNV [
18].
As part of the TRAC collaboration, 157 cryopreserved parasites were obtained from two sites in western Cambodia with a range of in vivo clearance phenotypes. Of these parasites, 68 were culture-adapted, and a sub-set of 36 parasites evaluated for their RSA0–3h phenotype. Using a high-resolution melt (HRM) genotyping assay for the most common pfkelch13 mutation (C580Y) and Sanger sequencing, pfkelch13 propeller mutations were tested for associations with both parasite in vivo clearance half-lives and in vitro parasite RSA0–3h survival rates. The analysis (1) identified parasites lacking pfkelch13 mutations, but exhibiting increased RSA0–3h survival phenotype (referred to as discordant parasites), suggesting that loci other than pfkelch13 may be involved in ART resistance; (2) demonstrated a new association between D584V and increased ring-stage survival; and, (3) detected a PPQ-resistant isolate among these parasites, consistent with other reports from this malaria-endemic region. Although there is a strong association between pfkelch13 mutations and increased ring-stage survival, mutations in pfkelch13 are not necessary for this ART resistance phenotype. Furthermore, in vitro PPQ resistance is present among these culture-adapted Cambodian parasites, which will enable further investigation of PPQ resistance mechanisms.
Methods
These parasite samples were obtained with informed consent from patients enrolled in the TRAC study in the Pailin and Pursat sites located in Western Cambodia. Full details of this study, the approvals, and the clinical and laboratory methodologies have been reported in detail elsewhere [
5].
Culture-adaptation and maintenance of TRAC parasites
All parasite samples were collected under protocols approved by ethical review boards in Cambodia, at Oxford University and at the Harvard T. H. Chan School of Public Health. Culture-adaptation of parasites was accomplished by thawing cryopreserved material containing infected red blood cells (iRBCs) that had been mixed with glycerolyte. Parasites were maintained in fresh human blood (O+) and Hepes buffered RPMI media containing 12.5% AB+ human serum (heat inactivated and pooled). Cultures were placed in modular incubators and gassed with 1% O
2/5% CO
2/balance N
2 gas and incubated with rotation (50 rpm) in a 37 °C incubator (Additional file
1).
Genetic material were extracted from filter papers (Whatman) and culture-adapted material using Promega DNA IQ Casework Pro Kit for Maxwell 16 (Promega Corp., Madison, WI, USA) and Qiagen (QIAmp DNA Blood Mini Kit) commercial kits, respectively, according to manufacturer instructions.
Genotyping
Development of a high-resolution melt (HRM) assay to screen populations for mutations around amino acid position 580 in the
pfkelch13 locus. The forward primer (5′-GGCACCTTTGAATACCC-3′), reverse primer (5′-CATTAGTTCCACCAATGACA-3′), and unlabeled, blocked probe (5′-AGCTATGTGTATTGCTTTTGAT-block-3′) were amplified asymmetrically at 0.5, 0.1, and 0.4 μM, respectively with 1 ng template DNA. After an initial 2-min hold at 95 °C, 5 or 10 μL reaction mixtures with 2.5 × HRM master mix (BioFire Defense, Salt Lake City, UT. USA) were PCR amplified for 55 cycles: 95 °C for 30 s, 66 °C for 30 s, and 74 °C for 30 s, followed by a pre-melt step of 95 and 28 °C for 30 s each. Products were melted from 45 to 90 °C on a BioFire Defense LightScanner-384 or -32 and analysed using the manufacturer software. Two plasmid controls containing the wild-type and mutant alleles were included as standards for every HRM run. Molecular barcoding was performed as described [
24,
25] to identify monogenomic samples with unique parasite genotypes.
Whole genome sequences
This publication uses sequencing data generated by the Pf3k project [
26]. The variant call files generated from this project were used to identify SNPs in each of the samples used in this study.
Pfkelch13 PCR sequencing strategy
The propeller domain of pfkelch13 was PCR amplified using Phusion HF DNA Polymerase kit and primers 3F and 1R′ in all 68 culture adapted parasites. DNA from KH001_024 was also amplified with primers 4F and 3R′. An aliquot of PCR product was resolved by gel electrophoresis to check for specificity and yield and the remaining product was purified using DNA Clean & Concentrator, ZymoResearch and sequenced using the same primers used to amplify the product by Genewiz. The full ORF of pfkelch13 was PCR amplified using primers 1F and 1R from 3D7 and individual culture-adapted parasites. The resulting PCR product of ~2.2 kb was purified by gel extraction (QIAquick Gel Extraction Kit, Qiagen) and sequenced at Genewiz using primers 1F, 2F, 3F, 1R, 2R and 3R. Primer sequences are as follows: 1F: 5′-ATGGAAGGAGAAAAAGTAAAAACAAAAGCAAATAG-3′; 2F: 5′-GGTAGGTGATTTAAGAATTACATTTATTAATTGGT-3′; 3F: 5′-CATTCCCATTAGTATTTTGTATAGGTG-3′; 4F: 5′-GTAGAGGTGGCACCTTTGAATACCCCTAGATCATC-3′ 1R: 5′-TTATATATTTGCTATTAAAACGGAGTGACCAAATCTG-3′; 1R′: 5′-TTA TAT ATT TGC TAT TAA AAC GGA GTG-3′; 2R: 5′-AGCCTTATAATCATAGTTATTACCACCAAAAACG-3′; 3R: 5′-TGTTGGTATTCATAATTGATGGAGAATTC-3′; 3R′: 5′-ATAAAATGTGCATGAAAATAAATATTAAAG-3′.
In vitro 72-h drug susceptibility by SYBR green staining
Drug susceptibility was measured using the SYBR Green I method as previously described [
27]. Briefly, tightly synchronized 0–6 h rings were grown for 72 h in the presence of different concentrations of drugs in 384-well plates at 1% haematocrit and 1% starting parasitaemia; and, growth at 72 h was measured by SYBR Green staining of parasite DNA. Except for PPQ and KH001_053 where a 24-point dilution series was used, a 12-point dilution series of each drug was carried out in triplicate and repeated with at least three biological replicates. DMSO stocks of drugs were dispensed by a HP D300 Digital Dispenser (Hewlett Packard Palo Alto, CA, USA) except for the CQ and PPQ stocks that were prepared in water and dispensed with a Velocity 11 Robot (Bravo). Relative fluorescence units (RFU) was measured at an excitation of 494 nm and emission of 530 nm on a SpectraMax M5 (Molecular Devices Sunnyvale, CA, USA) and analysed using GraphPad Prism version 5 (GraphPad Software La Jolla, CA. USA). EC
50 values were determined with the curve-fitting algorithm log(inhibitor) vs response—variable slope, except for PPQ and KH001_053. Due to the bimodal dose response of KH001_053 to PPQ, curve fitting didn’t give an accurate EC
50 value. The reported PPQ EC
50s for KH001_053 are estimates using biphasic curve fitting. Spearman correlation analysis was performed to assess the relationship between the anti-malarial EC
50 values and in vivo clearance half-life, ring survival assay value or
pfmdr1 copy number. p values <0.05 were considered significant.
Copy number variation assays
To determine copy numbers for
pfmdr1, plasmepsin II and the 63 kb amplicon genes (PF3D7_0520100, PF3D7_0520500, PF3D7_0520600, PF3D7_0520900 and PF3D7_0521000), real time quantitative PCR was performed on genomic DNA (extracted with QIAmp Blood Mini Kit, Qiagen) as previously described [
28] with the following modifications: Amplification reactions were done in MicroAmp 384-well plates in 10 μL SYBR Green master mix (Applied Biosystems), 150 nM of each forward and reverse primer and 0.4 ng template. Forty cycles were performed in the Applied Biosystems ViiA™ 7 Real-time PCR system (Life Technologies). Forward and reverse primers used were as previously described to amplify the following loci:
pfmdr1 (PF3D7_0523000) [
29], the 63 kb region on chromosome 5 (PF3D7_0520100, PF3D7_0520500, PF3D7_0520600, PF3D7_0520900 and PF3D7_0521000 [
20]) and
plasmepsin II (PF3D7_140800) [
22]. For the endogenous controls,
β-
tubulin forward and reverse primers [
28] were used for
pfmdr1, PF3D7_0520100 and PF3D7_0520900 while
pfldh forward and reverse primers [
30] were used for PF3D7_0520500, PF3D7_0520600 and PF3D7_0521000. Target primers used were validated to have the same PCR efficiencies as their endogenous control primers; and, average copy number values were calculated for each gene using data from three independent experiments.
Sequencing the pfcrt locus
The entire
pfcrt locus was sequenced as previously described [
20] with some modifications. Briefly, total RNA was extracted using RNeasy kit (Qiagen) and used to generate cDNA using Superscript III (Invitrogen). The resulting cDNA was then used as template for PCR amplification of
pfcrt [
20], followed by Sanger sequencing (GENEWIZ) [
20]. Sequence data analysis was performed using MacVector.
Ring survival assay (RSA0–3h)
The RSA
0–3h was performed as described previously [
6]. Essentially, parasites were sorbitol synchronized twice at 40-h intervals, synchronous 40–44 h segmented schizonts were incubated for 15 min at 37 °C in serum-free media supplemented with heparin to disrupt agglutinated erythrocytes and late stages were purified with 35/65% discontinuous Percoll gradient. The segmented schizonts were washed and cultured with fresh RBCs for 3 h, after which late stages were removed by sorbitol treatment. Cultures with 0–3 h rings were adjusted to 2% haematocrit and 1% parasitaemia and seeded into a 24-well plate with 1 ml complete media per well. To these wells, either DHA at 700 nM or 0.1% DMSO were added immediately and incubated for 6 h at 37 °C, washed and incubated in drug free media. At 72 h from seeding, thin blood smears were made from control and treated wells and survival rates were measured microscopically by counting the proportion of next generation viable rings with normal morphology. Survival rates were expressed as ratios of viable parasitaemias in DHA-exposed and DMSO-treated controls. Parasites were counted from 10,000 RBC, and two separate individuals served as independent slide readers.
Discussion
Within a set of 68 culture-adapted Cambodian parasites, some parasites exhibited an increased RSA0–3h phenotype that lack pfkelch13 mutations, a parasite harbouring the PfKelch13 D584V change exhibited increased RSA0–3h survival, and some parasites showed evidence for PPQ resistance. To evaluate a set of 157 Cambodian P. falciparum isolates from the TRAC study, with known clearance half-life data, a genotyping assay for the common C580Y change associated with ART resistance was developed and confirmed a clear association between delayed parasite clearance half-life and the presence of the C580Y allele. Sequence or genotype data from 146 of these parasites demonstrated a positive association between the clearance phenotype and pfkelch13 propeller mutations. 68 of these parasites were culture-adapted and 36 were tested for their in vitro RSA0–3h phenotype. Increased parasite clearance half-life was associated with RSA0–3h ≥1% values, with a 4-h or greater clearance phenotype being more consistent with ART resistance based upon RSA0–3h for this population. A novel positive association was demonstrated between D584V and in vitro ring-stage parasite survival under DHA among these parasites.
Despite this general correspondence between in vivo clearance and in vitro RSA
0–3h phenotypes and
Pfkelch13 propeller mutations, there were discordant parasites that exhibited a resistant RSA
0–3h phenotype (RSA
0–3h ≥0.8%) yet harboured no mutations in the entire
pfkelch13 ORF, consistent with other studies that found similarly discordant parasites by RSA
0–3h [
48], or by clearance half-life [
26]. A parasite (see Additional file
5) with a
PfKelch13 E270K outside the propeller domain exhibited a clearance value of >4 h, yet had an RSA
0–3h value of 0.4 %. These differences may be due to differential host factors including variances in drug pharmacokinetics, pharmacodynamics or host immune status. More isolates with an E270K mutation need to be studied to confirm the role of a
pfkelch13 mutation outside the propeller domain in ART resistance and reduce the probability of an artifact related to microscopy. Taken together, these results suggest that ART resistance in natural parasite populations could be mediated by changes outside of the
pkelch13 propeller domain. Parasites with these phenotypes around the defined cut off values may be important for identification and study of loci other than
pfkelch13 that contribute to ART resistance. Inspection of candidate loci, including traditional drug resistance mutations, recently identified secondary mutations, or loci related to pathways implicated in ART resistance, identified a few specific changes that may account for these discordant parasites, but lacked the statistical power to determine whether any of these contributed to ART resistance. However, identification of potential mutations in the
PIP5K and
pfmdr2 loci is consistent with other reports. Nevertheless, the identification of parasites that lack
pfkelch13 mutations yet harbour an increased RSA
0–3h survival phenotype, suggest loci other than
pfkelch13 may modulate ART resistance in these parasites. Alternative strategies, such as the use of independent chemogenomic strategies or genetic crosses in vitro [
49], might be useful to identify loci involved in conferring the observed increased ring-stage survival phenotype in the absence of
pfkelch13 mutations.
No relationship was found between EC
50 values for ART or its derivatives with either clearance or ring-stage survival phenotypes, but a previously noted correspondence was confirmed between
pfmdr1 copy number and EC
50 values of ART and AS, as well as with MFQ and LUM responses, among eight parasites evaluated for drug responses (Fig.
2). Drug testing identified a PPQ-resistant isolate among this population, consistent with reports in the literature of increased PPQ resistance in this region [
18,
19]. The PPQ-resistant parasite had a single copy of the
pfmdr1 gene [
20,
30] and amplification of the
plasmepsin II locus previously noted [
21,
22]. The exact nature of this resistance, the role of
plasmepsin II and the explanation for the apparent bimodal response to PPQ remains unknown and under investigation, but identification of culture-adapted parasites affords additional testing of both the phenotype and genotype (Additional file
1).
It has been suggested [
18,
19] that PPQ resistance arose in the context of or as a consequence of ART resistance. Understanding the nature of this resistance and the underlying mechanism will be critical for reducing or restricting emergence of PPQ resistance in Southeast Asia where reports indicate rapid emergence over the past several years [
18]. Furthermore, since DHA–PPQ is being utilized in mass drug administration campaigns designed to facilitate elimination of malaria in specific settings, use of molecular markers of resistance and understanding the relationship between ART and PPQ resistance will be important. Thus, identification of PPQ resistance with MFQ and LUM hyper-susceptibility in an ART resistant parasite could be an important clue to understanding mechanisms of drug response, and testing the implications of triple therapy, such as ART–PPQ–MFQ, being considered for use in this region [
18].
Conclusions
In a large set of P. falciparum isolates from the TRAC study, the associations between C580Y and several other pfkelch13 propeller mutations and parasite clearance half-life was investigated. In a subset of 68 culture-adapted parasites, RSA0–3h survival and conventional responses to multiple antimalarial drugs were measured. Several pfkelch13 mutations (including D584V) were associated with increased RSA0–3h survival, and discordant parasites with RSA0–3h survival 1% but without pfkelch13 ORF mutations were identified. These data suggest that mutations outside of Pfkelch13 may confer in vitro ART resistance in P. falciparum. It will therefore be important to continue phenotypic assessment of ART resistance, in addition to surveying for pfkelch13 propeller mutations. Detection of a PPQ-resistant parasite will enable further studies to investigate underlying mechanisms of PPQ resistance. This panel of culture-adapted parasites with known parasite clearance half-life, RSA0–3h survival, and pfkelch13 genotype will facilitate further investigation of ART resistance mechanisms, providing tools to identify potential PfKelch13-binding partners and other interacting molecules.
Authors’ contributions
AM, SB, PM carried out RSA0–3h, drug testing, PCR-based genotyping and copy number variation analysis and helped write the manuscript; WW carried out the genomic analysis with data provided by OM and helped write the manuscript; RD developed the C580Y HRM genotyping assay and carried out molecular barcoding of the samples; AD created primer sets and helped with RSA0–3h analysis of the samples; SS helped with genomic analysis and provided critical edits to the manuscript; CA, PL, MD, CW, EAA, AMD, NJW, and RF carried out field collection and provided in vivo sample information and samples. DFW provided critical review of the data and experimental guidance. SKV culture adapted the parasites, provided critical review of the data and experimental guidance, and wrote the manuscript. All authors provided critical review of the data and manuscript before publication. All authors read and approved the final manuscript.