Substantial reductions in malaria transmission, temporally associated with deployment of effective vector control and improved case management, have been observed in Africa during 2000–2010 (Noor et al., 2014; WHO, 2010). The considerable shift in epidemiology occurring in areas undergoing transition from malaria control to pre-elimination requires new tools and strategies for detection of malaria infections (Alonso and Tanner, 2013; Cotter et al., 2013; Tietje et al., 2014). The relative proportion of low-density infections that fall beneath the detection level of conventional microscopy and malaria rapid diagnostic tests (mRDTs) increases as transmission declines (Okell et al., 2009). These subpatent infections are likely to be asymptomatic and therefore missed by passive surveillance, but are estimated to potentially fuel 20–50% of human-to-mosquito transmission in pre-elimination settings (Okell et al., 2012). Low-density asymptomatic infections may therefore constitute an important reservoir for continued malaria transmission that needs to be targeted to achieve malaria elimination (Bousema et al., 2014).

Zanzibar was one of the first regions in sub-Saharan Africa to deploy artemisinin-based combination therapies (ACT) free of charge to all age groups through public health facilities. Artesunate–amodiaquine has been the first-line treatment for uncomplicated malaria in Zanzibar since September 2003. Deployment of artesunate–amodiaquine, together with mass distribution of long lasting insecticide-treated nets to high-risk groups, resulted in a substantial decline in Plasmodium falciparum malaria among febrile children, from approximately 30% to 1–2%, and a reduction of crude child mortality of approximately 50% between 2003 and 2006 (Bhattarai et al., 2007). LLINs were mass distributed in 2006 to high-risk groups, after which 90% of children under the age of five were reported to sleep under a LLIN (Bhattarai et al., 2007). Vector control was further strengthened by mass-distribution of two LLINs per household in 2008–2009, and annual rounds of IRS targeting all households in Zanzibar (excluding Stone Town) between 2006 and 2009 (Björkman et al. submitted). Coverage of vector control interventions remained high in 2009 despite reduced perceived threat of malaria among caretakers, with 70% of under-five children reported to sleep under a bed net and 94% living in a house targeted with IRS. Combined, 98% of children were covered by at least one of the vector control methods (Beer et al., 2013). Furthermore, mRDTs were introduced in 2006 for improved case detection, and targeting of treatment to patients with confirmed malaria infections. Malaria RDTs were shown to improve management of fever patients, providing adequate treatment and health outcomes without increased cost per patient (Msellem et al., 2009). Zanzibar is presently in a state of pre-elimination (Björkman et al. submitted), and the Zanzibar Ministry of Health has officially declared the aim of malaria elimination.

Deployment of ACT in Zanzibar has been a corner stone in the recent success in malaria control and monitoring continued efficacy is paramount 10 years after the introduction of artesunate–amodiaquine as first-line treatment. As resistance to ACT partner drugs has historically manifested before that of artemisinins (Venkatesan et al., 2014), molecular markers associated with amodiaquine resistance may serve as an important tool for surveillance of antimalarial drug resistance in Zanzibar (Froberg et al., 2012). Single nucleotide polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) transporter genes have been associated, both in vitro and in vivo, with resistance to amodiaquine (Echeverry et al., 2007; Folarin et al., 2011; Picot et al., 2009). Selection of pfcrt 76T and pfmdr1 86Y alleles, as well as pfmdr1 1246Y and the pfmdr1 (a.a.86,184,1246) YYY haplotype has been shown in recurrent infections after treatment with artesunate–amodiaquine or amodiaquine alone (Djimde et al., 2008; Duraisingh et al., 1997; Holmgren et al., 2006, 2007; Humphreys et al., 2007; Nsobya et al., 2007; Venkatesan et al., 2014).

The aim of this study was to describe temporal trends in the asymptomatic Plasmodium reservoir during the transition from high to low transmission in Zanzibar. Infections were characterised with regards to Plasmodium species, geographic- and age distribution, qPCR-determined parasite densities, complexity and diversity of infection, and temporal trends in P. falciparum SNPs associated with artesunate–amodiaquine resistance.

There is a declining, albeit persistent, reservoir of parasites present at low-densities in asymptomatic individuals in Zanzibar. The ability to accurately detect and subsequently treat all parasite carriers is fundamental for malaria elimination efforts.

As the prevalence of PCR-determined malaria declined in Zanzibar so did the relative proportion of infections that were detectable by microscopy or mRDT. High prevalence of sub-microscopic malaria has been reported in many other endemic areas (Ganguly et al., 2013; Golassa et al., 2013; Manjurano et al., 2011; Roper et al., 1996; Stresman et al., 2014). Okell et al. estimated that in low-transmission settings, where PCR-determined prevalence is less than 10%, microscopy would only detect on average 12% of all PCR-detectable infections (Okell et al., 2009, 2012). Our data confirm this and, importantly, our qPCR data showed that the majority of infections had densities much lower than the detection limits of both microscopy (50–100 p/μL) and mRDT (100 p/μL).

Several studies have demonstrated the transmission potential of low-density malaria infections (Bousema et al., 2014; Mawili-Mboumba et al., 2013), although their contribution to overall transmission has been debated (Lin et al., 2014). In Zanzibar, where it appears that subpatent infections constitute a large proportion of the parasite reservoir, it is important that infections are monitored using appropriate molecular tools (Cook et al., 2014). Despite the high sensitivity of PCR, infections with densities below or close to the PCR detection level are still missed. This limitation is also apparent in the low reproducibility of PCR-detectable low-density infections (Costa et al., 2014), and is important in discussions on the optimal strategy to clear remaining parasite reservoirs in malaria pre-elimination settings, by active infection detection or mass drug administration.

P. falciparum remained the predominant species throughout the study period. The prevalence was highest in older children and young adults (aged 5–25 years); with differential distribution between the two study districts. As malaria prevalence declines the epidemiology of malaria shifts, resulting in temporal heterogeneity and concentration of malaria in particular localities and demographic groups (Cotter et al., 2013). Characterisation of submicroscopic parasite populations requires highly sensitive molecular methods, but may provide important information for elimination programmes for scale-up and targeting of elimination strategies in remaining pockets of malaria infection (Tietje et al., 2014).

A significant reduction in the complexity of infection was observed between 2005 and 2009. MOI provides an alternative measure of transmission intensity (Stresman et al., 2014; Tusting et al., 2014), and our results correlate well with the reduction of transmission in Zanzibar. The heterozygosity, on the other hand, remained high over the study period with no significant difference between the years. The results are comparable to other geographical areas of sub-Saharan Africa (Anderson et al., 2000; Mwingira et al., 2011; Zhong et al., 2007), and suggest that the remaining parasite population is of high diversity. FST analysis showed no detectable reduction in the effective population size or bottlenecks during the transition from high to low transmission in Zanzibar. Similar findings have been reported from the highlands in western Kenya and Malawi (Sisya et al., 2015; Zhong et al., 2007), in contrast to the low genetic diversity and high genetic differentiation that has been observed in low transmission settings in parts of Asia and South America (Anderson et al., 2000). Changes in H_E occur slowly over time, and recent changes in malaria transmission combined with small sample sizes may therefore not provide the means to detect subtle changes in HE as a measure of genetic diversity (Sisya et al., 2015).

Artesunate–amodiaquine was deployed as a first-line treatment for uncomplicated malaria in all public health facilities in 2003. Intriguingly, a substantial decline in the pfcrt 76T and pfmdr1 86Y frequency occurred during the study period among PCR-determined parasite carriers, even though these SNPs have been associated, both in vitro and in vivo, with resistance to amodiaquine (Echeverry et al., 2007; Folarin et al., 2011; Picot et al., 2009; Venkatesan et al., 2014). A similar reduction was observed in febrile cases during the period 2002–2010 in Zanzibar (Froberg et al., 2012). These apparently controversial results may be attributed to the fitness costs associated with these SNPs (Babiker et al., 2009; Rosenthal, 2013). Resistance-mediating polymorphisms, that are associated with substantial fitness costs, result in the selection of wild-type alleles both in vitro and in areas of diminishing drug pressure (Babiker et al., 2009; Froberg et al., 2013; Rosenthal, 2013). A typical example is the return of the chloroquine sensitive form (pfcrt K76) in areas where chloroquine use has been discontinued due to high levels of resistance (Gharbi et al., 2013; Kublin et al., 2003; Ndiaye et al., 2012; Nkhoma et al., 2007; Wurtz et al., 2012). Genetic dilution by imported parasites from the African main-land could be another contributing factor to the decline in frequency since Artemether–lumefantrine, which selects in the opposite direction of artesunate–amodiaquine (pfcrt K76, pfmdr1 N86, 184F, D1246) (Venkatesan et al., 2014), is the first-line treatment in main-land Tanzania and Kenya. Importation of parasites may also explain the sustained high heterozygosity in Zanzibar (Zhong et al., 2007).

Interestingly, in the to-date most extensive report (a pooled analysis of individual patient data), none of the analysed pfcrt or pfmdr1 parasite genotypes were significant risk factors for recrudescent infections (treatment failures), but pfmdr1 86Y, 1246Y were selected in re-infections after treatment with artesunate–amodiaquine (Venkatesan et al., 2014). Furthermore, in patients treated with artesunate–amodiaquine, parasites carrying pfmdr1 86Y, 1246Y, or pfcrt 76T appeared earlier during follow-up than those carrying pfmdr1 N86, D1246, or pfcrt K76, indicating an advantage of harbouring these mutations when under direct drug pressure (Venkatesan et al., 2014). However, prolonged use of artesunate–amodiaquine does not seem to select for molecular markers that have been associated with amodiaquine resistance (Froberg et al., 2012), which is important for the continued efficacy of this ACT since treatment outcome is largely dependent on the long half-life partner drug (Venkatesan et al., 2014).

Frequencies of the pfmdr1 86Y, 1246Y SNPs and the pfmdr1 YYY and YYD haplotypes were significantly higher in asymptomatic infections than in symptomatic infections in 2013. Similar findings have recently been reported in Uganda and Benin, where the prevalence of wild-type genotypes were significantly higher in febrile children compared to asymptomatic children, suggesting greater virulence for wild type parasites (Ogouyemi-Hounto et al., 2013; Tukwasibwe et al., 2014). It could be that asymptomatic infections represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles and may therefore contribute to the epidemiology of drug resistant malaria (Brown et al., 2012). However, additional studies are required to determine if fitness is associated with virulence, and how it may influence onward transmission and spread of antimalarial drug resistance. Most likely these interactions are dependent on a complex interplay between, for example, parasite fitness, host immunity, transmission intensity, and drug pressure (Rosenthal, 2013).

There are several limitations with this study. Asymptomatic malaria was identified by PCR in healthy individuals participating in cross-sectional surveys. This study design gives a “snap-shot in time” and limits the understanding of the history, progression and dynamics of these infections. Importantly, reliable information on fever and treatments histories was not available for the study subjects, and the lack of gametocyte carriage data gives little insight regarding the contribution of these infections to malaria transmission. There are also several inherent limitations with PCR, such as the risk of false positivity and the limited sensitivity of RFLP assays to detect mixed infections. Finally, genotyping of molecular markers may be a useful tool for early detection of selection/development of resistance but should ideally be followed up by clinical trials to assess the true efficacy of artesunate–amodiaquine in Zanzibar.

There is a declining, albeit persistent, reservoir of parasites present at low-densities in asymptomatic individuals in Zanzibar. This study revealed important characteristics of the remaining parasite population, including intriguing temporal trends in molecular markers associated with antimalarial resistance, which need to be further investigated.