Background
Malaria is one of the most serious life-threatening protozoan diseases, classically identified by paroxysm, fever, and flu-like symptoms recurring in 48- or 72-h cycles. The World Health Organization (WHO) recommends artemisinin-based combination therapy (ACT) as the first-line treatment for falciparum malaria in all endemic regions [
1]. There is already evidence of reduced susceptibility of
P. falciparum to artemisinin derivatives in Southeast Asia which is manifested by the delayed parasite clearance times in vivo [
2,
3]. WHO has launched an action plan to stop possibly emerging resistance at an early stage so as to protect ACT [
4].
The
P. falciparum multidrug resistance 1 gene (
Pfmdr1),
P. falciparum chloroquine resistance transporter gene (
Pfcrt), and
kelch13 propeller region (
Pfk13) gene single nucleotide polymorphisms (SNPs) are believed to be markers of resistance to a great number of anti-malarial drugs, including ACT [
5,
6].
Increased copy number and sequence variation in
Pfmdr1 at the amino acid positions N86Y, S1034, N1042 and D1246Y have been reported to interfere with the parasite susceptibility to dihydroartemisinin [
7], artemisinin [
8,
9], mefloquine [
10], quinine [
8], halofantrine [
11], piperaquine [
9], lumefantrine [
12], amodiaquine [
13] and chloroquine [
14]. Recent in vitro work have strongly linked
Pfmdr1 to drug transportation [
15].
The
Pfcrt protein has capacity to efflux peptides and glutathione [
16], and transport anti-malarials besides chloroquine [
17]. Its codon K76T mutation has been linked to the development of chloroquine resistance [
18].
Pfcrt SNPs have also been found to be associated with the in vitro and/or in vivo parasite susceptibility to artemisinin [
19], lumefantrine [
20], quinine [
21], mefloquine [
18] and piperaquine [
22].
The
Pfk13 SNPs;C580Y, R539T, I543T, and Y493H were the four main alleles which were observed to be significantly involved in artemisinin resistance out of the 17 non-synonymous SNPs found in Cambodia [
5]. Other genes associated with artemisinin resistance include;
Pfcrt [
23]
, P. falciparum translationally controlled tumor protein (
Pftctp) gene [
24]
, Pfmdr1, P. falciparum multidrug resistance-associated protein-1 (
Pfmrp1) [
9] and ABC transporters [
25]. Also there are target proteins;
P. falciparum calcium adenosine triphosphatase 6 (
Pfatpase6) [
26] and
P. falciparum ubiquitin c-terminal hydrolase (
Pfubcth), the orthologue of
P. chabaudi ubiquitin protein-1 [
27].
The identification of the genetic determinants will be cornerstone for understanding the molecular basis of artemisinin drug resistance and will, therefore, provide basic evidence for molecular surveillance of drug-resistant P. falciparum strains for epidemiological surveys. This study reports an assessment of anti-malarial resistance marker polymorphisms in Pfmdr1, Pfcrt, and Pfk13 genes in chloroquine resistant W2 and chloroquine sensitive D6 lines cultured in presence of artemisinin.
Methods
Study site
The study was carried out at the Centre for Biotechnology Research and Development (CBRD) malaria laboratory, Kenya Medical Research Institute (KEMRI) Headquarters, Nairobi, Kenya.
Parasite cultures
Two
P. falciparum laboratory clones, W2 (Indochina) and D6 (Sierra Leone) were cultivated by standard methods, as described by Trager and Jensen [
28] with some modifications described by Benoit-Vical et al. [
29]. Continuous routine cultures were maintained at 1–5 % parasitaemia in complete medium containing 12.5 % heat inactivated human serum (blood type A, AB and O pooled) and in O
+ human erythrocytes. Haematocrit was adjusted to 6 % O
+ type RBCs of the 5 ml total culture volume. The choice of the two parasite lines (D6 and W2) was made in the anticipation that different genetic backgrounds of the parasites may provide a wider span of genetic determinants during the in vitro evolution by artemisinin selection.
Development of resistant parasite lines
Artemisinin resistance was induced in
P.
falciparum W2 and D6 lines as described by Oduola et al. [
30].
Plasmodium falciparum W2 and D6 lines were cultured under increasing artemisinin pressure in stepwise increments over 450 days. The starting concentration of artemisinin was 10 µg/ml for the two parasite clones (W2 and D6). Then, increments were in escalations of 10, 20, 30, 40 and 50 µg/ml, with an exposure time of 48 h at each increment.
The drug pressure cycle started with the addition of artemisinin to asynchronous cultures with 4–8 % parasitaemia. The 12.5 % human-serum-supplemented growth medium (RPMI Medium 1640; Gibco®), with or without artemisinin, was changed after 24 h. The artemisinin pressure was removed after 48 h, the time at which parasite morphology was degraded and growth was significantly reduced. The medium containing artemisinin was discarded and parasites washed twice with drug free wash media. Then, drug-free culture medium supplemented with 12.5 % human-serum was added to cultures, which were incubated at 37 °C until parasite growth and morphology became normal.
A parasite was considered “tolerant” to a particular artemisinin concentration when the recovery phase occurred within 48 h. The artemisinin exposure time was between 42 and 173 days so as to reduce the recovery time to below 48 h for the W2 and D6 lines at each increment. This was considered as a cycle of artemisinin pressure. At this point, artemisinin pressure was resumed with the consecutive higher artemisinin concentration in the increment to culture which has attained ≥5 % parasitaemia. During adaptation, parasite was frozen and the procedure was resumed from the cryopreserved stocks to maintain this phenotype.
Bulk culture of W2 cycle 5 culture A at 50 µg/ml artemisinin (W2 c5A), D6 cycle 3 culture A at 30 µg/ml artemisinin (D6 c3A) and D6 cycle 5 culture A at 50 µg/ml artemisinin (D6 c5A) parasites was cloned by limiting dilution for 35 days to give W2 cycle 5 sub-culture B (W2 c5B), D6 cycle 3 sub-culture B (D6 c3B) and D6 cycle 5 sub-culture B (D6 c5B), respectively. However, W2 cycle 3 culture A at 30 µg/ml artemisinin (W2 c3A) was not cloned. The samples of these cultures were collected and stored in extraction buffer for further analysis. Artemisinin together with other conventional anti-malarials [dihydroartemisinin (DHA), artemisinin (QHS), artesunate (AS), artemether (AM), chloroquine diphosphate (CQ), lumefantrine (LUM) and piperaquine phosphate (PIP)], susceptibilities were assayed by measuring the inhibition of [3H] hypoxanthine uptake in a sterile technique [
31], as described herein.
[3H] hypoxanthine incorporation assays
Plasmodium falciparum growth was determined by measuring incorporation of the nucleic acid precursor [3H] hypoxanthine in the parasites genomic DNA. Test compounds were diluted in DMSO (except CQ; diluted in water) and diluted to >0.2 % DMSO. 25 µl of screening medium was added to each well (except row B) in a 96-well flat-bottom micro-culture plate (Costar®, Corning Incorported, NY, USA). Fifty microlitre of dissolved compounds, containing four times the highest test concentration, were added to wells of row B in duplicates. Two times serial drug dilutions were prepared using a multichannel pipette. Twenty five microlitre of drug solution were taken from wells of row B and transferred, after mixing, to wells of row C and so forth down to row H (a 64-fold range dilution). The 25 µl of diluent removed from wells of row H were discarded. Two hundred microlitre of infected erythrocytes (1.5 % haematocrit and 0.5 % parasitaemia) were then added to each well except for wells A9–A12, to which, 200 µl of uninfected RBCs (diluted in screening medium to 1.5 % haematocrit) were added as a negative control.
The plates were incubated at 37 °C in a chamber gassed by a medical grade gas mixture of 92 % N2, 3 % O2, and 5 % CO2 at 95 % humidity. After a 48-h incubation period, 25 µl of [3H] hypoxanthine working solution was added by pulsing to each well (0.5 µCi per well).
Plates were incubated for an additional 18 h period then frozen at negative 20 °C. After thawing, the content of the plates was harvested onto glass-fiber filter mats (A 1450-421; Perkin Elmer) using a Betaplate cell harvester (1295-004 Betaplate; Wallac Perkin Elmer) and dried in an oven.
The oven-dried filter mats were drenched in 10 ml of scintillation fluid (Beta Scintilla®, Perkin Elmer, Schwerzenbach, CH) in a plastic foil (1450-432; Perkin Elmer) and the [3H] hypoxanthine incorporation was measured in terms of counts per minute (cpm) using a Betaplate liquid scintillation counter (1205 Betaplate; Wallac Perkin Elmer).
The result of each well was recorded as counts per minute and expressed as percentage of the untreated (positive) control (wells A1–A8; cultures without test compound). The negative control was used for background subtraction. IC50s (drug concentration at which 50 % of [3H] hypoxanthine incorporation was inhibited compared to drug-free controls) were estimated by linear interpolation [
32].
Data analysis was done using Graph Pad Prism software, version 5 (Graph Pad Software, Inc.), and the concentrations of the drugs that inhibited parasite growth by 50 % (IC50) were obtained by nonlinear regression analysis of the inhibition of uptake values and log-transformed concentration values.
Genomic DNA isolation
The genomic DNA was extracted from 1 to 2 ml aliquots of parasite cultures by using the QIAamp mini DNA kit (Qiagen, Germany) as per the manufacturer’s specifications and eluted in 50 µl of AE elution buffer. Non-template control samples were obtained by using un-parasitized red blood cells in each extraction. The untreated parasites (i.e., no drug pressure, W2 and D6) samples, cultured for the same period of 450 days, were used as the positive controls.
Sequencing of the Pfmdr1, Pfcrt and Pfk13 genes
The
Pfmdr1 (PFE1150w),
Pfcrt (MAL7P1.27), and
Pfk13 (PlasmoDB identifier PF3D7_1218300) genes were amplified from genomic DNA using polymerase chain reaction (PCR). The primers used for PCR and sequencing of the genes are shown in Table
1. The double-strand sequencing of PCR products was performed by Macrogen laboratory (Netherlands). Sequences were analysed by the BLAST programme in ncbi. Multiple nucleotide sequence alignment and analysis of the reported gene 3D7 in PlasmoDB DB and
Pfmdr1,
Pfcrt, and
Pfk13 sequenced
genes was done using the Codon code Aligner version 5.1 and MEGA 6 to identify specific single nucleotide polymorphism (SNP) combinations (standard bidirectional sequencing and reading).
Table 1
Primers used in PCR
Pfcrt MAL7P1.27 | 5 | P1 (5′-gcgcgcgcatggctcacgtttaggtggag-3′) | Forward | Primary PCR | _146 bp |
P2 (5′-gggcccggcggatgttacaaaactatagttacc-3′) | Reverse |
D1 (5′-tgtgctcatgtgtttaaactt-3′) | Forward | Nested PCR |
D2 (5′-caaaactatagttaccaattttg-3′) | Reverse |
Pfmdr1 PFE1150w | 7 | Af (5′-tgtatgtgctgtattatcaggaggaac-3′) | Forward | Primary PCR | _560 bp |
Ar (5′-aattgtactaaacctatagatactaatgataatattatagg-3′) | Reverse |
Bf (5′-gatggtaacctcagtatc-3′) | Forward | Nested PCR |
Br (5′-ctcctgataatacagcac-3′) | Reverse |
Pfk13 PlasmoDB PF3D7_1218300 | 13 | K13-1 (5′-cggagtgaccaaatctggga-3′) | Forward | Primary PCR | 845 bp |
K13-4 (5′-gggaatctggtggtaacagc-3′) | Reverse |
K13-2 (5′-gccaagctgccattcatttg -3′) | Forward | Nested PCR |
K13-3 (5′-gccttgttgaaagaagcaga -3′) | Reverse |
Nucleotide sequence accession numbers
The DNA sequences of representative samples showing mutant types were submitted to Gen-Bank and assigned accession numbers KU236713 and KU236714.
Discussion
There are several drawbacks to ACT as the current strategy, despite its success over the last decade. The WHO [
33] currently endorses five combinations artemether-lumefantrine, dihydroartemisinin–piperaquine phosphate, artesunate-amodiaquine, artesunate-mefloquine, and artesunate–pyronaridine. ACT is a highly used anti-malarial, creating a substantial selection advantage for parasites to evolve resistance independently with a precipitous decline in the artemisinin susceptibility.
In 2014, the number of courses treatment using various artemisinin-based combinations increased from 11 million in 2005 to 337 million [
34], but the mechanisms by which artemisinin act is still under investigation. Deducing molecular genetics on its resistance is paramount for determining treatment strategies, mapping the spread of resistance and guiding elimination [
27,
35,
36]. For the new tools for resistance surveillance to be validated a better understanding of the mechanisms of artemisinin resistance is needed. These tools are essential in guiding national treatment policies and helping in designing and deploying new drug combinations that may counter the emergence and spread of resistance.
In the present study, the discontinuous method of inducing resistance/tolerance to artemisinin in W2 and D6 lines of
P. falciparum was used [
30]. In agreement with other study [
36], the in vitro developed artemisinin-tolerant parasites could not grow permanently under artemisinin pressure. Hence resistance to artemisinin is different from resistance to other conventional anti-malarial drugs, such as amodiaquine. The culmination of in vitro resistance as used here in, is the ability for parasites to survive pharmacologically relevant exposure of artemisinin (levels considered clinically relevant in vivo) over many generations and by major increases in the IC50. After conducting the [3H] hypoxanthine incorporation susceptibility assays, it was found that resistant parasites showed the greatest tolerance for artemisinin (W2 c5B and D6 c5B). The study hypothesized a reduced susceptibility to this drug, because it was used to induce resistance in these strains. The discontinuous drug pressure selection scheme is supposedly to have been the key to success in selecting the tolerant lines [
30]. After adapting, the parasites were able to tolerate as much as 50 µg/ml of artemisinin for 48 h without noticeable changes in growth rate or morphology. The results also showed resistant parasites generally had decreased susceptibility to artemisinin, but were more susceptible to CQ, which is in agreement with other studies [
7,
37,
38]. There was a significant degree of cross-resistance to various other artemisinin derivatives, including artemether, artesunate, and dihydroartemisinin which were not utilized in the generation of resistance in those particular lines. This highlights that, it is possible to select resistance in a drug-sensitive background (D6) African isolate, in antagonism to conclusion from other studies in which it was stated that multiple resistance phenotypes must pre-exist [
39,
40].
These artemisinin-tolerant parasites provided a unique resource for the study of the artemisinin resistance mechanism(s) and modes of action based on analysis of SNPs in
Pfmdr1,
Pfk13 and haplotypes in
Pfcrt. The rationale for considering using these three genes were (i) that a relationship between SNPs in
Pfmdr1 and ACT has been reported, suggesting that
Pfmdr1-N86 or -184F was selected in reinfections after artemether-lumefantrine treatment; and moreover,
Pfcrt-76K and
Pfmdr1-N86 were selected by artemether-lumefantrine in recurrent infections [
20].
Pfmdr1 and
Pfcrt were selected for chloroquine resistance and they also both have been reported to be associated with in vitro reduced susceptibility to artesunate and amodiaquine [
41]. (ii) Genome association studies strongly suggested a locus on
P. falciparum chromosome 13 gene PF3D7_1343700 (bases 1,724,817–1,726,997, PlasmoDB 11.1) to artemisinin resistance and mutations discovered in the
Pfk13 were strongly linked to artemisinin resistance [
5].
It is well known that
Pfmdr1 is associated with drug resistance in Asia [
7,
38].
Pfmdr1 encode protein of the ATP binding cassette (ABC) transporter superfamily encoding P-glycoprotein homologue-1 [
42], which are localized to the parasite food vacuole membrane or plasma membrane and can decrease intracellular drug accumulation by pumping out drugs.
SNPs at codons 86, 184, and 1246 of the
Pfmdr1 gene has been suspected to be the markers of changes in parasite susceptibility to various drugs, including ACT [
43]. This shows that monitoring of changes in prevalence of SNPs in the
Pfmdr1 gene may serve as an early warning tool to selection of
P. falciparum tolerant/resistant to ACT [
44].
In the two artemisinin-tolerant parasite lines used in the present study, the decrease in parasite susceptibility to the drug was associated with substitution of mutation at codon Y184F for I185K. This conforms with other studies where sequence variation and increase in copy number in
Pfmdr1 were reported to interfere with the parasite susceptibility to dihydroartemisinin [
7] and artemisinin [
9]. This is because; the mutation at codon Y184F was not detected in the two studies. However, other studies have shown a relationship between SNPs in
Pfmdr1 and ACT drugs, in that
Pfmdr1-N86 or -184F was selected in reinfections and
Pfcrt-K76 and the
Pfmdr1-N86 in recurrent infections after artemether-lumefantrine treatment [
20].
It is established that ACT can select for particular alleles of two genes, Pfmdr1 and Pfcrt, which are firmly associated with resistance to the 4-aminoquinolines chloroquine and amodiaquine. The Pfcrt gene that encodes a 424-amino acid integral 10 trans-membrane domains protein is a transporter present in the parasite digestive vacuole membrane.
In this work, it was observed that
Pfcrt-CVMNK were the favoured haplotypes. This can be attributed to compensatory mutation at C350R in a different study, which was shown to explain the seemingly discordant chloroquine response in
Pfcrt-CVIET haplotype [
45]. This may suggest the benefit of the two wild-type genes,
Pfmdr1-N
86 Y
184 and
Pfcrt-CVMNK, was enhanced by the presence of artemisinin.
Ariey et al. [
5] linked artemisinin resistance in
P. falciparum to slow clearance rates in patients which he strongly associated with single point mutations in the “propeller” domain of the
P. falciparum kelch protein gene on chromosome 13 (
kelch13) (PlasmoDB identifier PF3D7_1218300). 17 mutations in the propeller region are linked to artemisinin drug resistance. The
Pfk13 gene is well conserved across
Plasmodium species and is thought to mediate protein–protein interactions [
5].WHO included
Pfk13 mutations (Y493H, I543T, R539T, C580Y and M476I) in the new working definition for suspected artemisinin resistance in August 2014 [
46]. The present study did not detect any polymorphisms reported elsewhere to reduce
P. falciparum susceptibility in vivo in the coding sequences of the
Pfk13 gene.
Although it is clear that
Pfmdr1 may play a significant role in artemisinin resistance, some studies also demonstrated that changes in susceptibility to mefloquine or halofantrine could be achieved without changes in the expression level or allelic type of
Pfmdr1 [
47,
48], suggesting a possible involvement of other genes and/or mechanisms of sustaining resistance in these parasites, including transient changes in gene expression.
Other work has reported lack of mutations in
Pfmdr1 and
Pfcrt in parasites cultured for 5 years under artemisinin derivatives in vitro, and
Pfmdr1 amplification was not observed. Further, four mutations were discovered in
Pfk13 (Y493H, I543T, R539T and C580Y). In the same study, 17 single K 13-propeller mutations were detected in naturally circulating parasites in Cambodia [
5]. However in the present study, there was
Pfmdr1 novel mutation at codon I185K and no non-synonymous mutations in
Pfk13 gene. This difference can be attributed to the shorter period (450 days) of artemisinin exposure.
Authors’ contributions
JNK was the principal investigator for the study, initiated, mapped the design, supervised and assisted in data analysis and manuscript review. FTK supervised the project activities, laboratory experiments and provided guidance on the analysis plan and manuscript review. DK initiated, mapped the designed, supervised and manuscript review. JK supervised the project; assisted in data analysis and manuscript review. MJN initiated, contributed to the study design, performed all experiments, analysed the data and wrote the manuscript. All authors read and approved the final manuscript.