Background
Falciparum malaria is still out of control, primarily because of the ability of the parasite to develop resistance against the used drugs. But, also the fast disseminations of the resistant parasites and possibly the accelerated ability of the parasite to develop resistance against new drugs, are important factors [
1]. Sulphadoxine/pyrimethamine (SP), has been an alternative to CQ for treatment and control of uncomplicated malaria in endemic countries, it was effective, affordable and complying drug. The fixed combination in SP inhibits the action of two enzymes, dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in folate metabolism pathway [
2,
3]. Mutations in the parasite genes coding for the two enzymes,
dhfr and
dhps, lead to SP resistance, however, the mutations also affect the parasite population, fitness and evolution [
4,
5].
Although elimination of the asexual stages of
Plasmodium falciparum is the focus of the treatment of individual symptomatic patients, at population level, reducing the carriage of gametocytes is necessary to limit the transmission of malaria parasites and the spread of anti-malarial drug resistance. In relation to SP treatment, the gametocyte carriage and infectivity to mosquitoes was found to be consistently higher in patients infected with drug resistant compared with drug sensitive malaria parasites [
6].
In malaria endemic areas, the host immune system acts synergistically with chemotherapy in the clearance of parasites [
7]. The susceptibility of parasites with mutations to the host immune mechanisms is largely unknown. In studies done in Mali, the term genotype-resistance index (GRI) was introduced to correct for the differences between the two arms of the equation; the treatment failure rate and the prevalence of parasite mutations [
8]. This index is a proxy for the role of immunity, which varies between epidemiological settings, and in the same setting depends on the age. The hypothesis on which this study was based was a combination of two old observations, the association of drug resistance with
dhfr/dhps mutation, and the implication of immunity in clearance of drug resistant parasites. That implies, the
dhfr/dhps mutant parasites might be more susceptible than the wild variants to host immune clearance. Further more the study aim to investigate the relationship between the
dhfr/dhps mutations and the parasite fitness in term of;
in-vivo parasite growth and gametocytogenesis. This data is complementary to a previous report in which these parameters were analysed in relation to
in-vivo drug response [
9].
Discussion
The mutations of genes encoding important enzymes such as DHFR and DHPS, should affects parasite metabolism and indeed change certain parasite characteristics. The goal of this study is to understand the implications of gene mutations of the two enzymes in certain parasite characteristics, namely the susceptibility of the parasite to immune clearance (parasite fitness), sexual reproduction (gametocytogenesis) and asexual reproduction (parasite growth).
The
dhfr/dhps gene mutations are known to be involved in parasite resistance to SP [
2,
3], and
in-vivo studies have shown that immunity is involved in clearance of drug resistant parasites [
9,
13]. Taken together, the
dhfr/dhps mutations are probably involved in immune clearance of SP resistant infections. In this study the ratio of TF to prevalence of the predominant quadruple
dhfr/dhps mutations (MOM 4) was approximately 1: 2, that was a circumstantial evidence for the role of host immunity in clearance of mutant parasites. The supporting evidence was the higher age (a surrogate marker for immunity) of the successfully treated patients. Interestingly, the ratio of MOMs prevalence to the actual treatment failure (2.03 and 2.74) is similar to the GRI (genotype resistance index) in Mali (1.6 to 2.8), a region epidemiologically similar to this study site [
14], although different drugs were used. The specific evidence for the increased suscebtibilty of the
dhfr/dhps multi-mutant parasites to immune clearance was the difference in age of the patients infected with parasites with the same number of mutations (MOM grade) but had different treatment outcome. This difference in age (ACR vs TF) was limited to the parasite infections with multiple mutations (MOM 2–5), but not to infections with wild parasites and parasites with single mutation (Figure
2B). For identification of the most important mutations (loci), larger sample size is needed, however, in this study, 51I, 108N, 437G and 540E were the predominant mutations (Figure
1). Furthermore, the reduced resistance of multi-mutant parasites to host immunity is a sign of reduced fitness, supporting previous reports [
4,
5,
15]. The degree of immunity which is required for clearance of parasite with specific number of mutation was estimated by the GRI value. The interpretation of this data indicates that, there are at least three factors directly contributing to parasite clearance (ACR): the drug (SP), immunity (represented by age) and parasite mutations (MOM > 1). The paradox is that, the
dhfr/dhps mutations provide the parasite the ability to resist chemotherapy, while it renders it more susceptible to the host immunity.
The pre-treatment parasite density probably reflects the
in-vivo parasite growth, assuming that sequestration is confined to severe malaria infection; and the peripheral parasitaemia represents the total parasite load. In this study, the parasite growth was influenced by
dhfr/dhps mutations where growth of wild and parasites with single mutation (MOM 1) was relatively higher from that of multi-mutant parasites in patients achieved ACR and the reverse was true in case of TF (Figure
2C). However, these results (trends) need to be taken with precaution because the sample size was small and the accuracy of the microscopic estimation of parasitaemia is not high, in addition parasite sequestration is not confined to severe malaria. In a previous study in the same site it was found that, there was significant association between reduced parasite growth and
dhfr/dhps mutations [
15].
It is known that, the frequency of gametocytaemia increases with treatment failure [
16], treatment with SP [
17] and younger age [
9]. In this study, the gametogenesis was significantly higher in parasites with multiple
dhfr/dhps mutations compared to wild type and parasites with single mutation, as recognized in other studies [
18,
19]. However, only two
dhfr/dhps mutations (MOM2) were needed to enhance the gametocytogenesis significantly, with no additive effect for more mutations (MOM 3–5). In the in-
vivo study, the patient achieved ACR had significantly lower frequency of gametocytaemia compared to patients who had TF. The association between gametocytogenesis and
dhfr/dhps mutations was independent of the treatment outcome and the host age. That is because, there was no association between the treatment outcome and the mutations in this study, and the age of patients infected with wild-type parasite and parasites with MOM1 (median 13 years) was not different from the age of patients who had TF (median, 12 years). Also, the differences cannot be due to gametocytogenic effect of SP treatment, as all patients were treated with SP. Although, the gametocytogenesis is the corner stone in the spread of the resistant strains, there are only few studies that had evoked the issue at the molecular level [
15,
19]. Finally, it is difficult to look at these parasite characteristics of gametocytogenesis, immune clearance and
in-vivo parasite growth in isolation of drug resistance since they are not independent from each other, thus, further studies are needed for detailed molecular exploration.
In conclusion, the dhfr/dhps gene mutations in P. falciparum are probably affecting the parasite fitness by rendering the parasite more susceptible to immune clearance than the wild-type variants. The increased immune clearance of mutant parasites can explain the lack of association between the prevalence of dhfr/dhps mutations and the TF rate in this setting. Furthermore, there were indications that the in-vivo asexual growth of the dhfr/dhps multi-mutant parasites was lower compared to wild types, while the gametocytogenesis in the former group was significantly higher than in the later. That is more likely an evolutionary mechanism compensating for the reduced asexual reproduction, and also can explain the fast propagation of SP resistance. Finally, the above-mentioned implications need only two (double) mutations in the dhfr/dhps genes irrespective of their codon/gene locus, while it was reported, SP resistance is associated with higher number of mutations in specific loci.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
IAE, MIE and HAG were contributed in all aspects of the study. IFK, MA and IB, were contributed in laboratory work, interpretation of results and paper writing. EM, contribution was basically in statistical analysis and paper writing