Plasmodium falciparum Na+/H+ Exchanger 1 Transporter Is Involved in Reduced Susceptibility to Quinine

Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC₅₀s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC₅₀ of QN (P = 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC₅₀ of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC₅₀ of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC₅₀ of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC₅₀ of QN (P = 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC₅₀ of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates.Malaria is the most important parasitic disease in the world, affecting 300 to 500 million people and killing 3 million people every year. Quinine (QN) has been used as a malaria treatment for more than 350 years in Africa, with little emergence and spread of resistance. QN remains the first-line antimalarial drug for the treatment of complicated malaria in Europe and Africa. However, despite QN′s efficacy against chloroquine-resistant strains, the emergence of QN resistance (QNR) has been documented. The first cases of QN clinical failure were observed in Brazil and Asia in the 1960s (4, 12). In the 1980s, clinical failures became more frequent in Southeast Asia, South America, and Africa (13, 15, 19, 22, 33). However, QNR is not yet a significant problem. QN remains the first-line drug for severe malaria and remains widely used at present as a second-line therapy for uncomplicated malaria in Africa and other areas. Artemisinin-based combination therapies were proposed as a first-line treatment for uncomplicated malaria 6 years ago. Since 2001, more than 56 countries have officially adopted artemisinin-based combination therapies for the treatment of Plasmodium falciparum malaria. However, individual P. falciparum isolates that are resistant to artemisinin in vitro in Cambodia have been described (14, 21). It is not clear whether these strains are associated with clinical failures. One strategy that health officials can pursue to reduce the prevalence of malaria is to combine QN with other antimalarial drugs such as tetracycline (8, 18) or clindamycin (16).Although some reports of treatment failure of QN exist, it is difficult to fully document QNR because of its short elimination half-life, the requirement to administer the drug three times a day for at least 5 days, drug intolerance often leading to poor compliance, and the lack of reliable data on the correlation between QN 50% inhibitory concentrations (IC₅₀s) and clinical failure. Maximizing the efficacy and longevity of QN as a tool for malaria control will depend critically on pursuing intensive research into identifying in vitro markers as well as implementing in vitro and in vivo surveillance programs such as those championed by the World Antimalarial Resistance Network (30, 31). In this context, there is a need to identify molecular markers that predict QNR and that can provide an active surveillance method to monitor temporal trends in parasite susceptibility (23).QNR appears to share common characteristics with chloroquine resistance. QNR is associated with mutations in both the P. falciparum multidrug resistance gene mdr1 (Pfmdr1) (20, 26) and the chloroquine resistance transporter gene Pfcrt (6, 7, 20). Nevertheless, the mechanism of QNR is still unclear. In addition to Pfmdr1 and Pfcrt, other genetic polymorphisms such as variations in microsatellite length on the sodium/hydrogen exchanger gene Pfnhe-1 (11) and mutations on the multidrug resistance protein gene Pfmrp might contribute to QNR (20). The evidence for the involvement of Pfnhe-1 or Pfmrp in QNR is limited. Only one previous study investigated the association of QN IC₅₀ and polymorphisms in the Pfnhe-1 gene in P. falciparum isolates (11).The objective of the present study was to investigate genetic polymorphisms in Pfcrt, Pfmrp, Pfmdr1, and Pfnhe-1 that could be associated with QNR in order to identify molecular markers of QNR that could be used for surveillance of resistance.