Background
Plasmodium falciparum malaria is a devastating disease still causing high mortality and morbidity especially in children in sub-Saharan Africa. The uncomplicated form of P. falciparum infection can be easily and successfully treated with an artemisinin-based combination therapy (ACT); however there is always the threat of resistance development to the different ACT compounds; the artemisinin derivative and/or the partner drug. Studies of P. falciparum polymorphisms associated with drug resistance can provide a useful tool to track resistance and guide treatment policy as well as an in-depth understanding of the development and spread of resistance.
Polymorphisms in
pfmdr1 and
pfcrt have been shown to have an effect on parasite susceptibility to artesunate–amodiaquine (AS–AQ) treatment, in particular
pfcrt 76T,
pfmdr1 1246Y and the
pfmdr1 86-184-1246 haplotype Y–Y–Y which has been associated with recrudescences and reinfections [
1‐
4].
Pfmdr1 N86, 184F and D1246 and
pfcrt K76 alleles are repeatedly demonstrated to be selected in reinfections or recurrent infections after artemether–lumefantrine (AL) treatment [
5,
6], supporting their role in the decreased sensitivity to lumefantrine. In pooled analyses, an increased risk of recrudescence after AL treatment was demonstrated when
pfmdr1 N86 was present [
4].
Only scarce data on anti-malarial efficacy and prevalence of molecular resistance markers is available from Liberia. In 1978–1981, wildtype alleles
pfcrt K76 and
pfmdr1N86 were dominating [
7]. In 2000, high clinical resistance to chloroquine was observed as well as high baseline prevalence of the chloroquine resistance marker
pfcrt 76T (84 %) [
8]. AS–AQ became the first line treatment in 2003 and it was changed to AS–AQ fixed dose combination (ASAQ-FDC) in 2010. High efficacy of ASAQ-FDC and AL was demonstrated in this clinical trial conducted in 2008–2009 [
9]. The aim of the study was to investigate the prevalence and selection of
pfcrt and
pfmdr1 genotypes in the clinical trial. This is the first study to assess molecular anti-malarial resistance markers in Liberia since the implementation of ACT. The work shows selection of parasite molecular markers in reinfections after treatment with both ACT and that molecular markers can influence the time after treatment and at which drug concentration a parasite is able to reinfect.
Discussion
Treatment with ASAQ-FDC and AL were highly effective against
P. falciparum malaria in this clinical trial. Recrudescences did not have a significantly lower concentration of lumefantrine or DAQ day 7 [
9]. Due to the low number of treatment failures, statistical analyses of the recrudescence genotypes were not meaningful. The number of reinfections was found to be high in the AL arm (30.0 %) and very high in the ASAQ-FDC arm (43.0 %) by day 42. In the ASAQ-FDC arm selection of the mutant allele
pfmdr1 1246Y and the haplotypes
pfmdr1 YYY and
pfmdr1 NYY in reinfections was demonstrated in Liberia, showing a similar selection as observed in Tanzania [
1] and in Mali [
2]. The baseline prevalence of
pfmdr1 1246Y at the time of this trial 2008–2009 was observed to be higher than in other countries in West Africa [38 % including mixed genotype infections compared to 0–25 % in 19 studies (search: years 2000–2014, n < 40, from West Africa)] as reported in literature [
10]. The high
pfmdr1 1246Y baseline prevalence may lead to the very high number of reinfections in the ASAQ-FDC arm in this trial, compared to the AL arm. Since 1246Y was observed to be selected in ASAQ-FDC reinfections, a higher baseline prevalence of the genotype can result in a larger pool of
pfmdr1 1246Y carrying parasites that can survive the residual DAQ levels and result in more reinfections. Selection of
pfmdr1 1246Y was observed mainly in early reinfections, while after day 35 the prevalence of 1246Y in reinfection was similar to the 1246Y prevalence before treatment. These results indicate that the concentration of DAQ was high enough to provide a selection pressure up to day 35. After this time-point all genotypes were able to survive the residual drug level and cause reinfection and there was no longer a protective or selective effect of the drug. This idea needs to be further elaborated by studying further efficacy trials with ASAQ and AL in West Africa. The 1246Y genotype has also been associated with ASAQ-FDC treatment failures [
4]. Despite the high prevalence of 1246Y, few treatment failures were observed in this study, probably due to the high efficacy of the artesunate compound in the combination. Artemisinin resistance is defined as delayed parasite clearance after treatment with an artemisinin or ACT [
11]. In this study a low proportion of patients were parasite positive on day 3 [
9], indicating high efficacy of the artesunate compound.
In the AL arm, the wild-type alleles
pfcrt K76,
pfmdr1 N86 and the
pfmdr1 haplotype NFD were selected in reinfecting parasites, by the drug pressure of residual levels of lumefantrine. Parasites harbouring
pfmdr1 N86 was not observed to reinfect significantly earlier, as has been suggested in previous studies [
4,
6]. However it was demonstrated that parasites carrying
pfmdr1 N86 could cause reinfection in patients with higher lumefantrine concentrations, than parasites carrying
pfmdr1 86Y. Patients with low lumefantrine levels could be reinfected with both N86 and 86Y alleles, while parasites with
pfmdr1 N86 could withstand intermediate drug concentrations. At high lumefantrine concentrations both genotypes would be killed and no reinfection could occur. This idea is supported by the observation that patients with reinfections had lower lumefantrine levels [
9]. This is in agreement with an important study suggesting that reinfecting parasites with
pfmdr1 N86 as well as the
pfmdr1 N86/184F/D1246 haplotype can withstand higher lumefantrine concentrations based on day 7 data [
12]. Both individual drug levels and the day of reinfection are key determinants for selection of reinfections and needs to be taken into account if available. This is especially important for AL-treated patients, in which the total lumefantrine dose received can vary significantly [
13]. In studies from East Africa
pfmdr1 184F is often selected in reinfections after AL treatment, which was not observed in this study. This could be due to the high baseline prevalence of
pfmdr1 NYD haplotype in this and a study in Benin [
3], where
pfmdr1 N86 could be the main driving force for lumefantrine resistance, independently of the
pfmdr1184 genotype.
The longer elimination half-life of the partner drug is a double-edged sword since although it can provide some post-prophylactic protection of individual patients, selection of reinfections can result in a more resistant parasite population over time. In high transmission areas, in Tanzania and Uganda, consistent treatment with AL overtime has probably resulted in significant increases in the prevalence of genotypes associated with AL treatment failure and reinfection [
14,
15]. However, temporal changes of ASAQ resistance markers have not been observed after consistent use of AS–AQ in Zanzibar [
16].
Conclusions
In this first study investigating pfcrt and pfmdr1 polymorphisms after the implementation of ACT in Liberia, selection of molecular markers in AL and ASAQ-FDC reinfections and high pfmdr1 1246Y baseline prevalence was demonstrated. The observation that parasites carrying pfmdr1 N86 can reinfect patients with higher lumefantrine concentrations highlights the importance of studying drug levels after AL treatment, as there could be large inter-individual dose variations. It is important to further investigate variables governing selection of reinfections in individual studies and as well as over time, as they can be responsible for the step from selection of a reinfection to full resistance and treatment failures. To evaluate and advise the current treatment policy it is essential to monitor temporal changes in molecular resistance markers of treatments used in Liberia in conjunction with conventional efficacy testing.
Authors’ contributions
SDO participated in the genetic analysis, performed the statistical analysis and drafted the paper, OMA participated in the genetic analysis, BS participated in the clinical trial, VJ participated in the drug concentration analysis, JJ participated in the clinical trial, YZ participated in the clinical trial, PH participated in the drug concentration analysis, EA participated in the conception of the clinical trial, JK participated in the conception of the clinical trial, PG participated in the conception of the clinical trial and the study, JLB participated in the conception of the study and participated in the coordination and the design of the study, SH participated in the conception of the study and participated in the coordination and the design of the study. All authors read and approved the final manuscript.