Analysis of the dihydrofolate reductase-thymidylate synthase gene sequences in Plasmodium vivax field isolates that failed chloroquine treatment

Background To use pyrimethamine as an alternative anti-malarial drug for chloroquine-resistant malaria parasites, it was necessary to determine the enzyme's genetic variation in dihydrofolate reductase-thymidylate syntase (DHFR-TS) among Korean strains. Methods Genetic variation of dhfr-ts genes of Plasmodium vivax clinical isolates from patients who did not respond to drug treatment (n = 11) in Korea were analysed. The genes were amplified using the polymerase chain reaction (PCR) with genomic DNA as a template. Results Sequence analysis showed that the open reading frame (ORF) of 1,857 nucleotides encoded a deduced protein of 618 amino acids (aa). Alignment with the DHFR-TS genes of other malaria parasites showed that a 231-residue DHFR domain and a 286-residue TS domain were seperated by a 101-aa linker region. This ORF shows 98.7% homology with the P. vivax Sal I strain (XM001615032) in the DHFR domain, 100% in the linker region and 99% in the TS domain. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed that nine isolates belonged to the sensitive strain, whereas two isolates met the criteria for resistance. In these two isolates, the amino acid at position 117 is changed from serine to asparagine (S117N). Additionally, all Korean isolates showed a deletion mutant of THGGDN in short tandem repetitive sequences between 88 and 106 amino acid. Conclusions These results suggest that sequence variations in the DHFR-TS represent the prevalence of antifolate-resistant P. vivax in Korea. Two of 11 isolates have the Ser to Asn mutation in codon 117, which is the major determinant of pyrimethamine resistance in P. vivax. Therefore, the introduction of pyrimethamine for the treatment of chloroquine-resistant vivax malaria as alternative drug in Korea should be seriously considered.


Background
Plasmodium vivax, a causative agent of relapsing benign tertian human malaria, is the second-most important human malaria and afflicts several hundred million people annually [1]. The disease is a major public health problem with associated socioeconomic ramifications for many temperate and most tropical countries.
Vivax malaria has been prevalent throughout the Korean peninsula for several centuries. However, as a result of a national malaria eradication program, and with help from the World Health Organization (WHO), the incidence of vivax malaria has rapidly decreased [2,3]. Vivax malaria was believed to have been eradicated in the Republic of Korea (ROK) since the late 1970 s, although two sporadic cases were detected in the 1980 s [4]. These two patients relapsed after a long incubation. In 1993, a vivax malaria case was diagnosed among ROK army soldiers serving in the northern Gyeonggi-do [5].
Chloroquine has been the standard treatment for blood-stage vivax malaria for over more than 40 years, whereas primaquine has been exploited for preventing relapses of the liver-stage hypnozoites. However, chloroquine-resistant P. vivax has been reported in several parts of the world [8][9][10][11][12][13], and relapse parasitaemia due to primaquine-resistant liver-stage hypnozoites has become increasingly widespread [14]. To manage chloroquine-resistant malaria, antifolate drugs including pyrimethamine, proguanil, and cycloguanil, in monotherapy and later in combination with sulphonamides, have been frequently employed as alternative anti-malarial agents in endemic areas. However, a few years after the introduction of antifolates to combat chloroquine resistance, clinical observations of slow parasite clearance and early reappearance of parasitemia after the therapy were reported, especially in P. vivax infections [15,16].
Pyrimethamine is an analogue of dihydrofolate that inhibits dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3). As in other protozoa, malarial DHFR is physically linked to thymidylate synthase (TS; EC 2.1.45), making DHFR-TS a bifunctional enzyme. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to inhibition of DNA replication [17]. Resistance to antifolate agents, possibly due to mutations at specific sites in DHFR, has been well studied in P. falciparum [18][19][20][21]. The loss of susceptibility to these antifolates results from mutations associated with reduced drug binding affinity to the active site of the DHFR enzyme [22]. A number of mutant forms of P. vivax dihydrofolate reductase (dhfr) have also been reported in various geographical regions [23][24][25]. These mutant dhfr of P. vivax encode amino acid sequences similar to those involved in antifolate resistance in P. falciparum and are responsible for antifolate resistance in a fashion similar to that previously reported in P. falciparum malaria [26,27].
Analysis of dhfr mutations in wild isolates has been considered as a valuable molecular approach for resistance mapping and the monitoring of malaria control measures [28,29]. In fact, the regimen of chloroquine (2,000 mg/3 days) and primaquine (15 mg/day for 14 days) has been used to treat vivax malaria patients since the re-emergence of P. vivax in Korea. However, the number of cases failing to respond to drug treatment has increased annually, and chloroquine-resistant malaria parasites have begun to spread nationally (Korea Centers for Disease Control and Prevention). Therefore, it is urgent that the dhfr-ts sequences be investigated before introducing antifolate drugs to control chloroquine-resistant malaria in Korea. Here, we first report the full gene sequences of DHFR-TS of P. vivax Korean isolates. These validations of molecular epidemiological efforts to study P. vivax in different endemic areas may be useful to establish an epidemiological map of drugresistant vivax malaria and to update anti-malarial policy guidelines in Korea.

Blood collection
Blood samples were collected from patients who experienced a relapse or re-infection. Thin and thick blood smears were prepared from patients' fingertips for microscopic diagnosis, after which additional blood samples were collected from the patients confirmed to be infected for further study. Informed consent was obtained from all patients. All samples were collected under human-use protocols that have been reviewed and approved by the Human Ethics Committee of the National Institute of Health in Korea.

Extraction of parasite DNA
Plasmodium vivax genomic DNA was extracted from the patients' blood using a QIAamp DNA Blood Kit (Qiagen, Valencia, USA) following the manufacturer's instructions. To confirm the diagnosis and to differentiate the infections caused by P. vivax, a single-step polymerase chain reaction (PCR) was performed as described previously [30].

Cloning and sequence analyses of the dhfr-ts gene
Polymerase chain reaction was done with P. vivax genomic DNA as a template, with specific primers for dhfr-ts to obtain the region between the start codon (ATG) and

Failed cases of chloroquine-primaquine combined treatment in Korea from 1996 to 2001
After a two-decades-long absence of vivax malaria in Korea, and starting with one patient who had served in the demilitarized zone (DMZ) as a soldier in 1993, the incidence of malaria increased rapidly [3][4][5][6][7]. To treat the vivax malaria patients, a combined chloroquine (2000 mg/3 days) and primaquine (15 mg/day for 14 days) regimen has been used as the standard treatment. Three years after the introduction of this standard regimen, in 1996, two cases failed to respond to the combination. Thereafter, 304 of 9,918 patents (3.07%) failed to be cured using this standard regimen (Table 1, Centers for Disease Control and Prevention, Korea).
Cloning of the dhfr -ts genes of P. vivax Korea isolates A total of 11 blood samples were collected from relapsed patients who visited the Public Health Care Centers located in Gyeonggido and Gangwondo in the malaria epidemic area of Korea. Microscopic examination followed by single-step PCR analysis revealed that all relapsed patients were infected with P. vivax. Several primers were synthesized -based on conserved regions in the DHFR and TS domains of the malarial DHFR-TS gene -to generate overlapping fragments of P. vivax genomic DNA. Full DHFR-TS gene PCR amplification products were obtained from P. vivax genomic DNA using the synthetic oligonucleotide primer pairs DHFR-F1 and DHFR-R. All of the PCR products were 1,857 bp. The complete DNA sequence of the DHFR-TS genes of P. vivax isolates were obtained by assembling the overlapping sequences produced using the DHFR-F1, DHFR-F2, DHFR-F3, and DHFR-R primers as described in Methods. To demonstrate that the four fragments were from the same gene, DHFR-F1 and DHFR-R primers hybridizing to the 5' and 3' ends, respectively, were synthesized to amplify, clone, and sequence the entire DHFR-TS gene. These primers included the methionine initiation codon and the stop codon. The combined nucleotide sequences from the PCR fragments contained a single open reading frame (ORF) of 1,857 bp encoding a deduced gene product of 618 aa. The nucleotide sequences encoding each gene were submitted to GenBank (Accession numbers; DQ514918, DQ514919, DQ514920, DQ514921, DQ517894, DQ517895, DQ517896, DQ517897, DQ517898, DQ517899 and DQ517900).
Comparison of the deduced DHFR-TS amino acid sequence of Korean P. vivax isolates with other P. vivax isolates When comparing the deduced amino acid sequences of DHFR-TS between P. vivax Sal I (XM001615032) and  [23]. The P. vivax dhfr gene contains a unique tandem repeat region between amino acids 88 and 106. Four allelic variants that differed with respect to the repeat motifs were previously identified [23]. The sequence THGGDN (18 bp) was missing in all Korean isolates ( Figure 4).

Comparison of the sequences from pyrimethaminesensitive and pymethamine-resistant parasites
The 12 non-synonymous point mutations in the dhfr domain of P. vivax have been reported [23,24,29]. Among them, mutations at codons 57 (F to L/I), 58 (S to R), and 117 (S to N/T) encode amino acids similar to those which cause antifolate resistance in P. falciparum, suggesting that antifolate resistance in P. vivax might have arisen in the same manner as that recognized in P. falciparum [24,27]. Nine of the studied Korean isolates had the wild-type genotype, whereas two isolates (KNIH98-55 and KNIH99-3170) had the single mutation S117N, which is key mutation associated with pyrimethamine resistance [17,27]. However, these resistant strains have amino acids similar to those in sensitive

Discussion
Only three years after the re-emergence of vivax malaria in Korea in 1993, it was revealed that the combined standard regimen of chloroquine and primaquine was not effective in all cases. Therefore, it is necessary to introduce another drug to control the chloroquine-resistant strains. One class of candidates is antifolate drugs, but strains resistant to this treatment have spread worldwide. To find a reliable treatment, it should be investigated the current status of antifolate drug resistance in vivax malaria.
Korean isolate (KNIH99-1063, DQ517897) shows 98.7% homology rate with P. vivax Sal I strain (XM001615032) in the DHFR domain, 100% in the linker region and 99% in the TS domain ( Figure 1). The TS domain shows high homology among different species (Pf, Pm, Po, Pb, and Pk; 87.3%-94.8%) (Figure 3), however low homology rates are observed in the linker  (2) is the deleted sequence from isolates PHI/ Djibouti and LGF/India. The latter isolates had a silent mutation at the nucleotide level (CAC CAG); (3) represents the deleted sequence of isolate VAN/Thailand and 11 Burmese isolates; (4) corresponds to the deleted sequence from isolate LFT/Cambodia. Two identical silent mutations (GGT GGC) and two mutations in 97 (Asn Lys) and 98 (Thr Arg) were detected [23]; (5) corresponds to the deleted sequence from Korean isolates (n = 11). Deletions are denoted by dashes. region (23.4% to 31.7%) (Figure 2). The DHFR domains show an intermediate homology rate, from 54.5% to 73.4% (Figure 1).
The relatively rapid propagation of antifolate-resistant P. vivax might be explained by this species' intrinsic resistance to antifolate drugs such as pyrimethamine. However, resistance acquired through sequential appearance of point mutations due to drug pressure and to the progressive selection of resistant parasites is a more plausible explanation for the failure of treatment of vivax malaria [31]. Molecular studies associated with clinical observations of vivax malaria have demonstrated that the major mechanism of antifolate resistance results from specific mutations in the dhfr gene of the parasite [24,29]. Although a total of 12 nonsynonymous mutations were found in P. vivax dhfr, based on secondarystructure analysis and homology modeling to P. falciparum dhfr, biochemical analysis of recombinant enzymes and clinical correlation demonstrated four point mutations at amino acid residues 57, 58, 61, and 117. These are thought to be critically involved in pyrimethamine resistance [24,27,29].
Two of 11 Korean isolates have the S117N single mutation. This single S117N mutation in PvDHFR conferred about 4000-and 1600-fold increased resistance to pyrimethamine and cycloguanil, respectively, compared to the wild-type PvDHFR [27]. The S58R/S117N double mutant PvDHFR was 10-to 25-fold less resistant than the S117N mutant to the inhibitors. Antifolate had never been used for the control of re-emerging vivax malaria in Korea, so we doubted about it. Interestingly, this single mutated allele (S117N) was identified at a high frequency in isolates in samples from Turkey and Azerbaijan (36% and 71% of isolates, respectively), areas where antifolate drug pressure of resistance is not obvious; this allele was not found in Indonesia (n = 36) or Thailand (n = 16). Additionally, it appears that P. vivax has developed resistance to sulfadoxine-pyrimethamine more rapidly than P. falciparum has; hence, artesunate plus sulfadoxine-pyrimethamine may not be effective overall against P. vivax in many areas [32].
The first-line treatment against P. vivax malaria in these areas is still the combination of chloroquine-primaquine, like in Korea, and a recent clinical study has demonstrated that treatment with chloroquine followed by treatment with primaquine is 100% effective in patients from Azerbaijan [33]. Therefore, it needs intensive molecular epidemiological studies in Korea with lots of samples. The easiest and most effective way might be to apply the restriction fragment length polymorphism (RFLP) analysis of the P. vivax DHFR gene as introduced by Imwong et al [24].
If the spread of chloroquine resistance encourages health care policy administators to move toward antifolate drug regimens as first-line treatment, data such as ours offer a snapshot of the prevalence of DHFR mutations in multiple geographical areas and can provide crucial information on the potential appearance of sulfadoxine-pyrimethamine resistance in Korea.

Conclusions
Together with these data, antifolate-resistant P. vivax is already starting to spread in Korea. Further detailed geographical mapping of current and changing patterns of drug resistance in vivax malaria on a national or regional scale would prove a valuable aid to developing and updating national anti-malarial policy guidelines in Korea. Control measures and inter-governmental cooperation are also needed to block the spread of drugresistant malaria in the country.