Status of potential Pf ATP6 molecular markers for artemisinin resistance in Suriname

Artemisia annua extracts have been used for centuries in traditional Chinese medicine, to treat febrile illnesses including malaria. Currently, artemisinin, isolated from the plant Artemisia annua and its synthetic derivatives are worldwide the most potent and rapidly acting anti-malarials. In 2003, the WHO recommended the use of artemisinin-based combination therapy (ACT) [1].

Parasite resistance, especially of Plasmodium falciparum, has been recorded to every utilized anti-malarial drug [2], which calls for a diligent monitoring of emerging artemisinin resistance in any malaria endemic area especially in light of the large parasite populations increasingly being exposed to artemisinins worldwide and the actual development of artemisinin resistance in Western Cambodia [3] and Thailand [4]. Various independent experimental approaches corroborate the emerging of artemisinin resistance: reports of cure rates below 75% after artemether-lumefantrine treatment in Cambodia [5]; the production of artemisinin-resistant strains in vivo in the rodent malaria model Plasmodium yoelii[6]; the selection of genetically stable and transmissible parasites resistant to artemisinin and artesunate in P. chabaudi chabaudi[7] and the observation of in vitro resistance to Artemether in isolates from French Guiana [8] and in travelers returning from sub-Saharan Africa [9].

Resistance to the anti-malarial drugs pyrimethamine, sulphadoxine and chloroquine is associated with specific mutations in the pfdhfr, pfdhps and pfcrt gene, respectively. However, there is considerable controversy on the notion of mutations responsible for the emerging resistance to the class of artemisinin derived anti-malarial drugs. Various individual molecules such as haem, catalase, SERCA type enzymes have been proposed as targets for artemisinins. Several observations suggest that artemisinins inhibit the P. falciparum sarco–endoplasmic reticulum Ca²⁺-ATPase (SERCA), encoded by the gene denoted PfATP6[10]. SERCA is a key metabolic enzyme, which reduces the cytosolic free calcium concentration.

Additional evidence that resistance to artemisinins may depend on single mutations in the SERCA-type Ca²⁺ ATPase was derived from a structure-function investigation, identifying a potential hydrophobic interaction between Leu₂₆₃ of malarial SERCA’s with a side chain of artemisinin derivatives [11] and several studies of in vitro resistance to Artemether. In Senegal, one isolate was a Pf ATPase6 E431K/A623E double mutant [8], in sub Saharan Africa, isolates were linked to the A623E/S769N haplotype [9] and in Suriname’s neighboring country French Guiana alongside the border Maroni river, an association with the S769N substitution was revealed [8]. On the other hand, in vivo artemisinin resistance could not be linked to mutations in the PfATP6 gene in clinical isolates from Cambodia [12].

The artemether-lumefantrine combination (Coartem®) was introduced in Suriname as the first-line treatment of malaria in January 2004 as part of an elaborate effort to strengthen the national malaria programme.

An active trans-border migration, especially of gold miners with high mobility and frequent use of counterfeit drugs, exists between Suriname and neighboring French Guiana, where in vitro resistance to Artemether was observed [8]. Although no in vivo resistance to artemisinin derivatives or ACT had been reported from French Guiana and artemisinin resistance was not yet clinically documented in Suriname, the circumstances called for an investigation of the potential emergence of artemisinin resistance in accordance with the WHO recommendations ‘to take urgent action to protect the efficacy of ACT’ [13].

A retrospective molecular survey was conducted to assess the status of three possible PfATP6 molecular markers for artemisinin resistance in clinical isolates collected in the three leading endemic regions in Suriname, including the border region with French Guiana. Parasites from two different time periods in Suriname were used to gain insight in the status of the PfATP6 gene prior to and after the introduction of ACT as first line anti-malarial drug. Furthermore, these polymorphisms of the PfATP6 gene were analyzed in paired isolates from patients with recurring malaria after controlled ACT therapy.

Polymorphisms in the PfATP6 gene, encoding the SERCA-type Plasmodium falciparum sarco–endoplasmic reticulum Ca²⁺-ATPase, were assessed in 28 samples derived from patients with microscopically confirmed uncomplicated P. falciparum malaria, from three different endemic regions.

Sufficient DNA was recovered for all 28 samples and single nucleotide polymorphic assays were successfully performed for all three amino acid positions 263, 623 and 769.

The four paired samples of patients with recurring malaria after controlled treatment were all indeed recrudescent malaria isolates, as was demonstrated with repeat number and recrudescence analysis.

Position Leu₂₆₃, was unaltered in all 28 investigated samples and none of the isolates displayed the A623E mutant genotype or the S769N mutation. It should be noted that even the recrudescent samples obtained in the border area with French Guiana still exhibited the wild-type S769 haplotype.

All 28 isolates exhibited an identical genotype for the three investigated positions. No changes could be detected either in samples obtained from geographical different regions or in samples collected prior to or after adopting an ACT regimen. However, all results should be regarded as indications since the sample size was small and an 18-24 months period is rather brief to exert selection pressure. The absence of the S769N mutation in clinical isolates corroborates the finding from neighboring Brazil, suggesting that the mutant S769N haplotype has probably not yet spread regionally [20].

An important finding is the observed in vivo decreased artemisinin susceptibility, as attested by recrudescent malaria despite monitored treatment, independent of the A623E, L263E and S769N mutations. Although the sample size was small, the results do not favor a direct association between these single nucleotide polymorphisms and resistance to artemisinin, which is supported by research on the P. chabaudi chabaudi ATP6 sequence revealing no nucleotide changes following selection with artesunate or artemisinin [7].

These results are also consistent with independent investigations conducted in Tanzania [21] and other African [22] and Asian [12] countries, where polymorphisms of PfATP6 could not be linked to artemisinin resistance, but are in contrast with the findings in travelers returning from sub-Saharan Africa [9], where the A623E/S769N haplotype was linked to in vitro Artemether resistance. Although several samples were collected alongside the Maroni border river and even with the recurring malaria isolates originating from this area, the results from neighboring French Guiana where the S769N mutation was reported to be associated with in vitro Artemether resistance [8], could not be substantiated.

The finding that generation of artemisinin resistant P. chabaudi chabaudi clones failed in chloroquine sensitive clones and was only successful in chloroquine resistant clones [23] did not confound the results, since all investigated clones were resistant to chloroquine [Adhin, unpublished results].

In the light of accumulating evidence of artemisinin resistance slowly emerging worldwide, diligent surveillance is needed to monitor susceptibility to artemisinin derivatives in endemic areas and these results underscore the search for more appropriate molecular markers. The determination of pfmdr1 copy number and single nucleotide polymorphisms in pfmdr1 alongside PfATP6 analysis might be helpful, since conflicting reports exist about the association of increased pfmdr1 copy number and reduced susceptibility not only to Mefloquine, but also to artemisinin [9, 12, 24]. The use of other screening methods as monitoring parasitaemia should be considered.

This report presents the first data for Suriname on single nucleotide PfATP6 polymorphisms, potentially associated with artemisinin resistance. Isolates obtained prior to or after adopting Coartem® as first-line regimen do not exhibit any mutations at the investigated polymorphisms since all the investigated samples from Suriname harbor the wild-type genotype at all three positions; L263E, A623E, S769N. The findings in French Guiana regarding the S769N mutation could not be substantiated, despite the geographic vicinity. The occurrence of malaria relapses after controlled ACT without altering the Pf ATP6 genotype at the investigated positions, proved the possibility of decrease of artemisinin susceptibility independent of these mutations in the PfATP6 gene.