Polymorphisms in Plasmodium vivax antifolate resistance markers in Afghanistan between 2007 and 2017

Background Plasmodium vivax is the predominant Plasmodium species in Afghanistan. National guidelines recommend the combination of chloroquine and primaquine (CQ-PQ) for radical treatment of P. vivax malaria. Artesunate in combination with the antifolates sulfadoxine-pyrimethamine (SP) has been first-line treatment for uncomplicated falciparum malaria until 2016. Although SP has been the recommended treatment for falciparum and not vivax malaria, exposure of the P. vivax parasite population to SP might still have been quite extensive because of community based management of malaria. The change in the P. vivax antifolate resistance markers between 2007 and 2017 were investigated. Methods Dried blood spots were collected (n = 185) from confirmed P. vivax patients in five malaria-endemic areas of Afghanistan bordering Tajikistan, Turkmenistan and Pakistan, including Takhar, Faryab, Laghman, Nangarhar, and Kunar, in 2007, 2010 and 2017. Semi-nested PCR, RFLP and nucleotide sequencing were used to assess the pyrimethamine resistant related mutations in P. vivax dihydrofolate reductase (pvdhfr I13L, P33L, N50I, F57L, S58R, T61I, S93H, S117N, I173L) and the sulfonamide resistance related mutations in P. vivax dihydropteroate synthase (pvdhps A383G, A553G). Results In the 185 samples genotyped for pvdhfr and pvdhps mutations, 11 distinct haplotypes were observed, which evolved over time. In 2007, wild type pvdhfr and pvdhps were the most frequent haplotype in all study sites (81%, 80/99). However, in 2017, the frequency of the wild-type was reduced to 36%, (21/58; p value ≤ 0.001), with an increase in frequency of the double mutant pvdhfr and pvdhps haplotype S58RS117N (21%, 12/58), and the single pvdhfr mutant haplotype S117N (14%, 8/58). Triple and quadruple mutations were not found. In addition, pvdhfr mutations at position N50I (7%, 13/185) and the novel mutation S93H (6%, 11/185) were observed. Based on in silico protein modelling and molecular docking, the pvdhfr N50I mutation is expected to affect only moderately pyrimethamine binding, whereas the S93H mutation does not. Conclusions In the course of ten years, there has been a strong increase in the frequency pyrimethamine resistance related mutations in pvdhfr in the P. vivax population in Afghanistan, although triple and quadruple mutations conferring high grade resistance were not observed. This suggests relatively low drug pressure from SP on the P. vivax parasite population in the study areas. The impact of two newly identified mutations in the pvdhfr gene on pyrimethamine resistance needs further investigation.


Background
With a sharp decline in falciparum malaria, Plasmodium vivax has become the prominent Plasmodium species in Afghanistan causing more than 95% of all malaria cases [1]. The recommended first-line treatment for vivax malaria in Afghanistan is chloroquine combined with primaquine for radical cure (CQ-PQ), whereas for uncomplicated falciparum malaria the combination of artesunate and sulfadoxine-pyrimethamine (AS-SP) has been first-line treatment from 2003 to 2016, replaced by artemether-lumefantrine since 2016. Although SP has been the recommended treatment for falciparum and not vivax malaria, exposure of the P. vivax parasite population to SP might still have been quite extensive because of community based management of malaria particularly symptom-based clinical or probable diagnosed malaria in past two decades [2,3] and SP treatment of vivax malaria in parts of the private sector or through self-medication [3]. This might have increased antifolate resistance in P. vivax in Afghanistan.
Dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) are 2 essential enzymes in de novo folate synthesis pathway in Plasmodium and the drug targets of pyrimethamine, and sulfadoxine, respectively [4]. SP is considered a sub-optimal treatment for P. vivax infections, because this parasite is intrinsically less sensitive to sulfonamides and resistance to SP is rapidly acquired with extensive drug exposure [5]. Through clinical, epidemiological, molecular and biochemical studies it has been identified that SP resistance in P. vivax is conferred by specific point mutations in the P. vivax dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) genes. In areas with extensive SP use, treatment failure of vivax malaria with SP rapidly evolved, and was associated with mutations in codons 57, 58, 61, 117, 173 of pvdhfr and in codons 382, 383, and 553 of pvdhps [6][7][8][9][10].
In this study, the frequency of SP resistance related point mutations in the P. vivax pvdhfr and pvdhps genes in Afghanistan over time between 2007 and 2017 were reported. In addition, in silico three-dimensional modelling and molecular docking of 2 newly identified pvdhfr mutationswere performed to predict their impact on pyrimethamine sensitivity.

Study sites and sample collection
Dried blood spots (n = 185) from patients with light microscopy confirmed P. vivax infection were collected from five malaria endemic provinces in Afghanistan. In 2007 samples were collected from Takhar (bordering Tajikistan), Faryab (bordering Turkmenistan), and Nangarhar (bordering Pakistan). In 2010, samples were collected from Kunar and in 2017 from Nangarhar and Laghman, all bordering Pakistan. Blood samples of 20-30 µl were collected onto filter paper at the moment of patient presentation before antimalarial treatment. Genomic DNA was extracted by using QIAmp ® DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions and purified DNA was stored at − 20 °C until further processing. Approval for this study was obtained from the Ethics Review Committee for Research in Human Subjects, Faculty of Tropical Medicine, Mahidol University, Thailand (EC approval number MUTM 2019-065-01).
In this study, the frequency of mixed infection of falciparum and vivax malaria was investigated by nested PCR detected 18S rRNA of Plasmodium spp. [32], only one sample was P. vivax and P. falciparum mixed infection (0.54%, 1/185). The low frequency of mixed infection of P. vivax and P. falciparum was explained by P. falciparum infected samples were excluded in the beginning.

Pvdhfr and pvdhps amplification and genotyping
Amplification of pvdhfr by semi-nested Polymerase Chain Reaction was performed following established and published methods protocols [8,23]. For amplification, the primary and secondary reaction volumes were 25 and 100 µl, respectively, including 1 µl of template genomic DNA added in the primary amplified reaction, and 3 µl of primary amplified product added to the second round of parasite population in the study areas. The impact of two newly identified mutations in the pvdhfr gene on pyrimethamine resistance needs further investigation.
Keywords: Plasmodium vivax, Antifolate resistance, Dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), Afghanistan amplification. The reaction mixture contained a final concentration of 125 nM primers forward-reverse mixture, 10 mM Tris-HCL (pH 8.3), 2 mM MgCl2, 125 µM dNTP mixture, and 0.4 U Taq polymerase (Invitrogen, Carlsbad, CA) (Additional file 1: Table S1-S7). The DNA fragments from PCR amplification or Restriction Fragment Length Polymorphism (New England BioLabs Inc., Ipswich, MA) were identified by 3% metaphor agarose gel electrophoresis (Radnor Corporate Center Radnor, PA). The amplified PCR product of pvdhfr was purified for DNA sequencing using PCR purification kit, FavorPrep ™ (Favorgen, Taiwan). The purified PCR products of pvdhfr were sequenced by Macrogen, Korea. Nucleotide and amino acid sequences of this gene were aligned and compared with the P. vivax reference sequence from the original Sal1 strain (accession no. XM001615032), using BioEdit v7.2.5. For identification of established gene mutations associated with sulfonamide resistance, genotyping of pvdhps used PCR-RFLP methods, following established and published protocols [33]. The positive control of P. vivax is genomic DNA from SalI reference strain and characterized P. vivax isolates from the patient. To ensure the accuracy of the results, two positive controls and negative controls were added for quality control at every step of the procedure.

In silico modelling of mutant PvDHFR
A computer simulated three-dimensional model of the PvDHFR protein structure and its putative interactions with pyrimethamine was used to predict the impact of pvdhfr mutations on pyrimethamine binding. The wildtype PvDHFR structure (PDB ID: 2BL9) was used as a template for mutant PvDHFR modelling. The model was constructed using SWISS-MODEL (https ://swiss model .expas y.org). The constructed model was verified by PROCHECK [34]. Then, the derived structures of the active sites of mutant PvDHFR were complexed with pyrimethamine and evaluated by AutoDock Vina [35]. The structural models were visualized by Discovery Studio Visualizer-Accelrys.

Statistical analysis
The data of SNPs frequency related drug resistant genes were analysed by using MS Excel and SPSS v26.0. IBM Corp., Armonk, NY, USA. Pearson's Chi-square test was used to compare proportions of haplotypes. A P value < 0.05 was considered statistically significant.
A novel mutation at codon S93H (AGC to CAC) was identified comprising a single mutant haplotype S93H (4%, 8/185) and a double mutant haplotype S93HS117N (2%, 3/185) ( Table 1). The N50I mutation (AAC to ATC) observed in this study had not been described from Afghanistan before. The mutation was part of the single N50I mutation haplotype present in 2% (3/185) of samples. While double mutant haplotype N50IS117N was found in 5% (10/185) of samples. Haplotypes with triple or quadruple mutations, which are associated with high grade antifolate resistance, were not observed in this study.

In silico modelling of novel pvdhfr mutations
Since the two nonsynonymous pvdhfr mutation at position N50I, AAC (Asn) to ATC (Ile), and S93H, AGC (Ser) to CAC (His) had not been observed earlier in Afghanistan, the corresponding three-dimensional structures of the mutated proteins and the impact on pyrimethamine binding were modelled and predicted. The mutation N50I is located within the binding pocket, approximately 5 Å from the pyrimethamine molecule but does not directly interact with the drug. The mutation S93H is placed on the other side of the protein molecule, far away from the binding pocket (Figs. 3,4,5). Replacement of Table 1  Asn with Ile at position 50 disrupts the hydrogen bonds between Asn and water molecules. The N50I mutation also causes a conformation rearrangement of the helix (residues 50-63), disturbing the favorable interactions between the enzyme and inhibitor. The model predicted that the pvdhfr N50I mutation would affect only moderately pyrimethamine binding, whereas the S93H mutation would not affect binding.

Discussion
The combination of AS-SP has been used as first-line treatment for uncomplicated.
P. falciparum in Afghanistan since 2003 to 2016, and SP has been widely available in private sectors for a long time. Since an episode of falciparum malaria triggers relapse infection with P. vivax a few weeks later, this provides a window of selection for resistant parasites to outgrow sensitive parasite still killed by the remaining SP drug levels. Drug pressure from SP on the P. vivax parasite population over the past decade is thus likely, which might cause gene mutations in pvdhfr and pvdhps causing reduced SP sensitivity. Indeed, in this study, the frequency of the double mutant pvdhfr haplotype at S58RS117N and single mutant haplotype at S117N had increased significantly over the course of ten years since 2007 were observed. However, the triple and quadruple haplotype mutations associated with higher level of antifolate resistance were not found, suggesting only low or moderate drug pressure from SP on the P. vivax parasite populations in the study areas.
These findings agree with previously published reports showing low prevalence (less than 5%) of triple and quadruple mutant pvdhfr and pvdhps haplotypes in P. vivax parasite populations from Iran, Pakistan and India (Table 2) [21,22,[26][27][28][29][30][31][36][37][38][39]. In the wider study region, it was observed that the pyrimethamine resistance marker at position 117 in pvdhfr arose first and was followed by the subsequent mutation in positions 117 and 58, conferring increasing levels of resistance as report earlier [23,[26][27][28][29][30][31]. Antifolate drugs inhibit two essential enzymes in the folate biosynthesis pathway, necessary for DNA precursor synthesis. Sulfadoxine inhibits DHPS while pyrimethamine inhibits DHFR, which results in parasite death [4]. In P. falciparum, the intensive use of SP as first line treatment in south-east Asia since 1970s resulted in rapid acquisition of mutations in pfdhfr and pfdhps, causing amino acid changes in the enzymes at positions in  the binding pockets for pyrimethamine and sulfadoxine, respectively, causing SP resistance [7,8]. The antifolate resistant mechanism in P. vivax, with incremental resistance acquired by the accumulation of single point mutations in pvdhfr and pvdhps, is thought to be similar to the antifolate resistance mechanism in P. falciparum. In P. vivax, the sequence of non-synonymous mutations in pvdhfr is in codons 13, 58, 117, 173, equivalent to the orthologous pfdhfr positions 16, 59, 108 and 164 in P. falciparum. In P. vivax, biochemical and protein functional studies showed that pyrimethamine effectively inhibits wild-type PvDHFR, but were approximately 60 to > 4000 times less active against mutant enzymes. Double mutant S58R and S117N PvDHFR was 10-25 fold less inhibited by pyrimethamine than the S117N mutant [10]. This relates to the steric hindering of the pyrimethamine binding pocket caused by the S117N mutation in the PvDHFR enzyme [9,10].The studies also showed that a change in the effect of pyrimethamine on the enzyme kinetic properties of mutant PvDHFR is closely related with a change in sensitivity to pyrimethamine [9,10]. The current study identified a novel nonsynonymous mutation S93H in pvdhfr and for Afghanistan newly observed N50I mutation in pvdhfr. The N50I mutation corresponds to the orthologous mutation N51I in P. falciparum. The pvdhfr N50I mutation was previously observed in low frequency in Pakistan [29]. Previous study shown that mutant N50I PvDHFR expressed in yeast confers an increase in pyrimethamine IC 50 compared to wild type PvDHFR, whereas, the combination of mutation N50I and S117N conferred a-57 fold increase in IC 50 compared to wild type [40]. To assess the interaction of these mutations on pyrimethamine binding, homology modelling and molecular docking in-silico were performed. This showed that the pvdhfr N50I mutation interrupted moderately the binding of pyrimethamine. In contrast, the pvdhfr S93H mutation, changing amino acids from AGC (Ser) to CAC (His) located away from the binding pocket of pyrimethamine, was predicted not to interrupt pyrimethamine binding. However, these in silico results will need confirmation in further in vitro or in vivo drug sensitivity studies.

A I P N F R T S N I / G
An additional marker for SP resistance in P. falciparum is increased copies number of GTP cyclohydrolase I gene (pfgch1). GTP cyclohydrolase is another important enzyme in folate biosynthesis, and an increased copy number might compensate for the fitness loss associated with triple and quadruple mutations in pfdhfr and pfdhps [41,42]. So far, there is limited number of reports on gene amplification of pvgch1, therefore still no finding of the association of mutations in pvdhfr/pvdhps and pvgch1 copy number variation. Whether copy number variation (CNV) in the P. vivax orthologous gene pvgch1 is important in antifolate resistance needs further study. Pvgch1 CNV was not assessed in the current study, but less likely to be present because of the absence of triple-or quadruple mutations in pvdhfr in our samples.

Conclusions
This study shows that although wild-type pvdhfr and pvdhps haplotypes have become less frequent in the Afghanistan P. vivax parasite population between 2007 and 2017, but only double and single nonsynonymous mutations in pvdhfr were observed, which would confer only moderate SP resistance. This suggests relatively low drug pressure from SP on the P. vivax parasite population in the study areas. Of the two novel pvdhfr mutations identified in Afghanistan, only the N50I was predicted to moderately affect pyrimethamine binding.