Background
Sulfadoxine–pyrimethamine (SP), or Fansidar, a combination anti-malarial drug containing the sulfonamide antibiotic, sulfadoxine and the antiprotozoal pyrimethamine, has long been used as an anti-malarial drug throughout the world. Its easy single-dose prescription and relative efficacy has made
this combination an excellent choice in the treatment of uncomplicated falciparum malaria and intermittent presumptive treatment in pregnancy (IPTp) in Africa with the advent of chloroquine resistance [
1]. Nevertheless, the growing parasite resistance to this drug has limited its use in many parts of the world, including Indonesia.
The molecular basis of parasite resistance to SP has been established in
Plasmodium falciparum and rodent
Plasmodium, and various single nucleotide polymorphisms (SNPs) in dihydropteroate synthase (
dhps) and dihydrofolate reductase (
dhfr) genes have been linked to the resistance [
2,
3]. The SP combination has never been recommended to treat vivax malaria in Indonesia, however, the sympatric existence of
P. falciparum and
Plasmodium vivax and the practice of malaria treatment without microscopic confirmation suggest that accidental treatment of vivax malaria with SP has often taken place. Treatment of patients with SP has inadvertently led to the simultaneous selection of SP-resistant
P. vivax. In Indonesia before 2004, SP was used alone as a second-line anti-malarial drug for falciparum malaria [
4]. Although SP has never been recommended for the treatment of patients with
P. vivax malaria, the selection pressure exerted by the drug is expected to have continued progressively in
P. falciparum and
P. vivax [
5]. Since 2010, vivax malaria cases in Indonesia are treated with artemisinin-based combination therapy (ACT) [
6].
Pyrimethamine inhibits the
dhfr enzyme [
7] and sulfadoxine targets the
dhps enzyme in the folate biosynthetic pathway of the parasite [
8]. Point mutations in parasite
dhfr and
dhps genes confer resistance to SP in
P. falciparum. High level resistance to pyrimethamine in
P. falciparum results from the accumulation of mutations in
pfdhfr principally at codons 16, 51, 59, 108, and 164 [
9,
10]. These mutations have been shown to alter the pyrimethamine binding sites in
pfdhfr and reduce enzyme drug interaction [
11]. Twenty non-synonymous mutations have already been described in the
pvdhfr gene [
5,
12]. Some of these mutations (at codon 57, 58, 61, 117, and 173) are involved in resistance to pyrimethamine [
13,
14]. Five mutations have already been identified in the
pvdhps gene, at codon 382, 383, 512, 553, and 585, corresponding to position 436, 437, 540, 581, and 613 of the homologues gene in the
P. falciparum [
3,
14,
15]. The
pvdhfr and
pvdhps genotypes might be associated with treatment failure in individual vivax malaria patients [
16]. Limited data are available about polymorphisms in
pvdhfr and
pvdhps genes of malaria parasites from Indonesia. Previous data from Lampung show triple mutation found in this area [
17] and a quadruple mutant 49R/57L/58R/61M/117T was found in Papua [
17,
18].
The extent of genetic polymorphisms associated with resistance to SP was screened among the P. vivax isolates in Indonesia towards evaluating the use of SP for the treatment of P. vivax malaria and the possible use of SP for IPTp.
Discussion
Analysis of the
pvdhfr and
pvdhps genes in
P. vivax isolates from five different malaria-endemic areas in Indonesia revealed a wide distribution of the mutant alleles associated with resistance to SP. The mutant alleles of the
dhfr gene were found either as single polymorphisms or in combination with other polymorphisms. With the
dhps gene, 383G was the only mutant allele observed. Previous reports found similar polymorphisms in
P. vivax isolates from Lampung and Papua [
17,
18]. The findings indicate that the SP drug pressure to
P. vivax may have taken place at all sites and that mistreatment of
P. vivax malaria infections may be widespread. Until 2010, the recommended drug for vivax malaria was the combination of chloroquine with primaquine but due to the spread of chloroquine resistance to
P. falciparum and
P. vivax, ACT was introduced [
24].
Three types of allelic combination of pvdhfr gene were observed among the P. vivax isolates from the five malaria-endemic areas investigated. These included double, triple and quadruple mutants. Overall, when looking at the allelic combinations, the frequency of double mutants was 22.6 % (36 isolates), triple mutants was 2.5 % (four isolates) and quadruple mutants was 15.1 % (24 isolates). Plasmodium vivax isolates from Papua were dominated by quadruple mutants (61.8 %). Nevertheless, the in vivo study in Sumba to determine the molecular basis of SP treatment failure in P. vivax indicated that all P. vivax isolates that carried the aforementioned allelic combination were still susceptible to SP, except in one recurrent isolate at day 14 with the double dhfr mutants, 58R/61M.
Mutations in
pvdhfr and dhps genes, including 58R and 117N, have been implicated in pyrimethamine and sulfadoxine resistance, respectively [
25]. In
P. falciparum, the existence of quintuple mutations, three in
dhfr gene (S118N, C59R and N51C) and two in
dhps gene (A437G and K540E), have been associated with SP treatment failure [
26‐
28]. The corresponding mutations in
P. vivax are 117N, 58R and 49R in
dhfr gene and 383G and 553G in
dhps gene. Although
dhfr mutations have been widespread among the
P. vivax isolates, mutations at the
dhps gene are still rare. The 383G allele was found in various frequency among the isolates examined and the highest frequency was observed in Purworejo.
Two and three GGDN repeat units were detected in wild and mutant types
dhfr in all allelic combination. This result corroborates the previous results where an insertion/deletion event within the short repetitive region did not appear to be clearly associated with antifolate resistance. However, repetitive sequences that produce length polymorphism may not affect pyrimethamine sensitivity [
24].
The wide distribution of
dhfr and
dhps gene polymorphisms among the
P. vivax field isolates seems to have little implication on the efficacy of SP combination treatment. This was demonstrated in the in vivo study in west Sumba District, where the efficacy of SP was still very high (94 %). This is the first report from Indonesia that demonstrates that
P. vivax is highly sensitive to SP. The results of this study however require further observation in areas where SP was reported to be resistant to
P. falciparum, such as Papua [
18]. Unfortunately, with current policy that adopted ACT as first-line therapy for both
P. vivax and
P. falciparum, it will be difficult to justify a study to monitor the efficacy of SP anymore. Nevertheless, SP may be considered as a rational option for the treatment of vivax malaria either given alone or in combination with artemisinin. Previous studies in Pakistan and Afghanistan also found high efficacy of SP in the treatment of vivax malaria [
29].
Most subjects carried triple and quadruple mutations in the
dhfr gene in addition to the mutaion in
dhps 383G, but still were sensitive to the SP. The only subject who showed late treatment failure carried the double mutations in
dhfr without any
dhps gene mutation. Here, it is not clear as to whether this parasite originated from new infection or was a recurrence from the current infection, keeping in mind that previous results indicate that hypnozoites may have a different genotype than that of the initial infection [
30,
31]. Nonetheless, as SP has a long half-life, any parasite that was detected during the monitored 42-day treatment should be relatively resistant to SP. Previous results from Thailand also show inconclusive results upon the association between the
dhfr and
dhps mutations to the SP treatment outcome. Although it is clear that the isolates that carry multiple mutations in
dhfr and
dhps are associated with high grade SP resistance, many of the isolates still respond adequately to the SP [
23]. In this regard, it is important to note that in Indonesia, the proportion of
P. vivax isolates that carry double mutant
dhps is still very rare and in fact was only reported in the isolates collected from northeastern Papua. Papua, with the highest amount of malaria and consequent highest selection pressure from mistreatment of vivax infections with SP, may explain this high number of multiple resistant alleles. These results may explain why the
P. vivax isolates in Sumba are still susceptible to SP and this also may be true of the isolates from Lampung, Purworejo, Mataram and, to a lesser extent, in Papua.
The development and spread of drug-resistant parasite strains is a major obstacle to the malaria control and elimination programme. As the molecular basis of the parasite resistance to antifolates and sulfa drugs has been well established in Plasmodium spp., analysis on the frequency distribution of dhfr and dhps mutant alleles would provide a better perspective on the use of SP in a particular area. In this regard, it is important to notice an increasing prevalence of P. vivax isolates carrying the triple mutants of dhfr gene in all sites and particularly the quadruple mutants of dhfr gene in northeastern Papua regions.
Authors’ contributions
PBSA and DS conceived the study, and participated in its design and drafted the first manuscript. PBSA, SSM, RN, WS, RMD, S, AST, and M assisted in field work. IER was engaged in data collection, cleaning, analysis and contributed to data interpretation. PBSA, SSM and RN performed the PCR with input from PBSA. PBSA and DS supervised the laboratory procedures and PCR quality control. DS, NFL and RWS oversaw the study design and provided input to data analysis, interpretation and helped check the draft manuscript. All authors contributed to submitted version of the manuscript. All authors read and approved the final manuscript.