Decreased prevalence of Plasmodium falciparum resistance markers to amodiaquine despite its wide scale use as ACT partner drug in Zanzibar

Background Zanzibar has recently undergone a rapid decline in Plasmodium falciparum transmission following combined malaria control interventions with artemisinin-based combination therapy (ACT) and integrated vector control. Artesunate-amodiaquine (ASAQ) was implemented as first-line treatment for uncomplicated P. falciparum malaria in Zanzibar in 2003. Resistance to amodiaquine has been associated with the single nucleotide polymorphism (SNP) alleles pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y. An accumulation of these SNP alleles in the parasite population over time might threaten ASAQ efficacy. The aim of this study was to assess whether prolonged use of ASAQ as first-line anti-malarial treatment selects for P. falciparum SNPs associated with resistance to the ACT partner drug amodiaquine. Methods The individual as well as the combined SNP allele prevalence were compared in pre-treatment blood samples from patients with uncomplicated P. falciparum malaria enrolled in clinical trials conducted just prior to the introduction of ASAQ in 2002–2003 (n = 208) and seven years after wide scale use of ASAQ in 2010 (n = 122). Results There was a statistically significant decrease of pfcrt 76T (96–63%), pfmdr1 86Y (75–52%), 184Y (83–72%), 1246Y (28–16%) and the most common haplotypes pfcrt/pfmdr1 TYYD (46–26%) and TYYY (17–8%), while an increase of pfcrt/pfmdr1 KNFD (0.4–14%) and KNYD (1–12%). Conclusions This is the first observation of a decreased prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y in an African setting after several years of extensive ASAQ use as first-line treatment for uncomplicated malaria. This may support sustained efficacy of ASAQ on Zanzibar, although it was unexpected considering that all these SNPs have previously been associated with amodiaquine resistance. The underlying factors of these results are unclear. Genetic dilution by imported P. falciparum parasites from mainland Tanzania, a de-selection by artesunate per se and/or an associated fitness cost might represent contributing factors. More detailed studies on temporal trends of molecular markers associated with amodiaquine resistance are required to improve the understanding of this observation.


Background
Zanzibar has recently undergone a rapid decline in Plasmodium falciparum transmission following combined malaria control interventions with artemisinin-based combination therapy (ACT) and integrated vector control [1,2]. In the new epidemiological context, where in vivo trials to assess ACT efficacy have been increasingly difficult to conduct due to limited number of patients, surveillance of molecular markers associated with anti-malarial drug resistance may be useful as an early warning system of development and spread of ACT resistance.
Artesunate (AS) plus amodiaquine (AQ) combination therapy (ASAQ) was implemented as first-line treatment for uncomplicated P. falciparum malaria free of charge to all age groups through public health care facilities in Zanzibar in September 2003. AQ and its slowly eliminated active metabolite desethylamodiaquine (DEAQ) are 4-aminoquinolines and structurally related to chloroquine (CQ). Despite the similarities and putative crossresistance in between the compounds, AQ/DEAQ has remained more efficacious [3,4].
The aim of this study was to assess whether prolonged use of ASAQ as first-line anti-malarial treatment selects for P. falciparum SNPs associated with resistance to the ACT partner drug AQ.

Methods
The prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y were compared in pre-treatment blood samples collected on filter papers (3MM W , Whatman, UK). Samples were collected from individuals with uncomplicated P. falciparum malaria, residing in North A (Unguja Island) and Micheweni (Pemba Island) districts in Zanzibar. Patients were enrolled in clinical trials conducted just prior to the introduction of ASAQ in 2002-2003 (n = 208) [16,17] and seven years after wide scale use of ASAQ in 2010 (n = 122) (Shakely et al. 2012, unpublished data). Malaria diagnosis was confirmed by blood smear microscopy and rapid malaria diagnostic (RDT), respectively.
A mixed infection was considered to contain two P. falciparum strains, contributing with one of each SNP alleles during PCR-RFLP. In the haplotype analyses all isolates including mixed SNP results at more than one position were excluded. Allele and haplotype prevalences between 2002-2003 and 2010 were compared by chi square tests (SigmaPlot W 11.0, Systat Software Inc, USA). Statistical significance was defined as p < 0.05.

Discussion
This is the first observation of a decreased prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y in an African setting after several years of extensive ASAQ use as first-line treatment for uncomplicated malaria. This may support sustained efficacy of ASAQ on Zanzibar, although it was unexpected considering that all these SNPs have previously been associated with AQ/DEAQ resistance.
The underlying factors of these results are unclear. Genetic dilution by imported P. falciparum parasites from for example mainland Tanzania could represent a contributing factor. Even though Zanzibar is a part of Tanzania, they are independent in some issues e.g. the malaria control programme. Mainland Tanzania implemented artemether-lumefantrine (Coartem W ) as first-line treatment in 2006 when this ACT was widely manufactured, price had reduced and studies were shown it was safe to give children below ten kg. Artemether-lumefantrine, has shown to select for the opposite alleles i.e. pfcrt 76K, pfmdr1 86N, 184F and 1246D [21][22][23][24].
Another contributing factor may be that AS per se potentially selects for pfcrt 76K, pfmdr1 86N and 1246D, which have been associated with decreased susceptibility to the artemisinins in vitro [25,26]. Importantly however, no such selection has been shown after monotherapy with artemisinin derivatives in vivo.
A third contributing factor may be that SNPs associated with AQ resistance cause a fitness cost to the parasite, which would affect the selection pattern under different drug pressures. In competition experiments between modified isogenic clones, only differing in the pfmdr1 1246 position, pfmdr1 1246Y was found to be associated with a substantial fitness cost to the parasite (Fröberg et al. 2012, unpublished data). This could also  apply on the other SNPs and also explain the haplotype results in this study. Before ASAQ implementation the most common haplotype was TYYD, indicating that the previous first-line treatment i.e. CQ mainly selected for pfcrt 76T, pfmdr1 86Y and 184Y. The second most common haplotype was TYYY, where pfmdr1 1246Y has mainly been associated with AQ/DEAQ resistance. Seven years later a significant selection of KNFD and KNYD was observed. Hence, the individual SNPs pfcrt 76T, pfmdr1 86Y and 1246Y rarely exist alone, suggesting that they may be associated with a significant fitness cost and support each other in a possibly synergistic and/or compensatory relationship, whereas pfmdr1 184Y do exist alone and might not largely affect fitness.
Finally, even though these SNPs have been selected for after AQ/ASAQ treatment, the association with AQ/ DEAQ resistance may not be that strong that it will spread with prolonged wide-scale use of ASAQ.

Conclusions
Seven years after wide scale use of ASAQ as first-line treatment in Zanzibar, SNPs associated with AQ/DEAQ resistance have not been selected for. Instead, the prevalence of these SNPs has decreased, which may support sustained efficacy of this ACT as first-line treatment in Zanzibar. However, the results were unexpected, which calls for more detailed studies of temporal trends of molecular markers associated with AQ/DEAQ resistance both among symptomatic and asymptomatic P. falciparum infections to improve the understanding of this observation.