Background
Artemisinin-based combination therapy (ACT) has been the recommended treatment against uncomplicated malaria caused by
Plasmodium falciparum since 2002 [
1]. It is also often used against other
Plasmodium species. In 2016, over 400 million doses were distributed, with the large majority being administered in Africa [
2]. ACT has made a significant contribution to the reduction in malaria cases observed between 2000 and 2015, supporting their important role in malaria control [
3]. ACT quickly reduces malaria symptoms and densities of asexual malaria parasites [
4], while it can also reduce gametocyte carriage and malaria transmissibility compared to non-ACT medicines [
5‐
8]. A recent meta-analysis concluded that the most widely used ACT medicine, artemether–lumefantrine (AL), is superior to non-ACT medicines in reducing gametocytaemia and malaria transmission [
9]. Nevertheless, ACT has a limited effect against mature gametocytes, and gametocytes can still be transmitted from
P. falciparum-infected patients following ACT [
10‐
14].
Gametocytes are transmitted to mosquito females when they take a blood meal from infectious humans, and, after parasite development in infected mosquitoes, malaria parasites are transmitted to humans in the form of sporozoites, again during blood feeding.
Anopheles gambiae sensu lato (s.l.). and
Anopheles funestus s.l. are the most important vector species in sub-Saharan Africa [
15], partially due to their strong preference for blood feeding on humans [
16]. To find a host, mosquito females use volatile cues emanating from the skin, besides CO
2 that is associated with the presence of all vertebrate hosts. In
An. gambiae sensu stricto (s.s.) and
Anopheles coluzzii, siblings within the
An. gambiae species complex, anthropophilic biting behaviour is associated with a strong preference for human skin odour over skin odour of other vertebrate host species [e.g.
17].
Surprisingly, there are no studies on the possible effects of ACT on malaria-transmitting mosquitoes. Yet, it is important to investigate such effects because transmission of gametocytes to mosquitoes is still possible after ACT as described above [e.g.
14]. The rate of post-treatment transmission could be influenced if ACT has an effect on the attractiveness of treated individuals and consequently on the probability of contact between ACT-treated humans and mosquitoes. Several factors are known to affect human attractiveness to mosquitoes through effects on skin odour composition, including infection with malaria parasites [
18]. Here, the effect of AL on the attractiveness of parasite-free humans is assessed by studying mosquito responses towards their skin odour.
The rate of malaria parasite transmission could also be affected by ACT, if ACT would directly affect the lifespan of infected mosquitoes and consequently the probability of parasites within the mosquito reaching the sporozoite stage. Female mosquito fitness was therefore studied when females take a blood-meal supplemented with AL. Although
Plasmodium infection may affect mosquito survival [e.g.
19], and the effects of ACT may be different in infected mosquitoes, uninfected mosquitoes were used in this study as a first investigation of the potential endectocidal effects of ACT against
Anopheles.
Discussion
Malaria mosquitoes rely on skin odour to find their human host [e.g.
31], and many factors contribute to interindividual variation in attractiveness of healthy humans [
25]. Here, the influence of anti-malarial treatment with ACT on human attractiveness to malaria mosquitoes was investigated. This is relevant because ACT has a limited effect against gametocytes, which can lead to post-treatment gametocytaemia and transmission to mosquitoes. A recent meta-analysis showed that approximately 25% of patients that report with gametocytes, may still carry gametocytes 1 week after AL-treatment, the most common form of ACT currently used in Africa [
14]. This proportion gradually decreases over the course of the next few weeks. Several studies demonstrated that post-treatment gametocytaemia can indeed result in transmission to mosquitoes 1 week after anti-malarial treatment with ACT [
13,
32]. Skin odour samples collected from healthy participants on the final day of a three-day administration course with AL had the same attractiveness as skin odour samples collected before AL-administration. This was shown in a dual-port olfactometer in the laboratory with
An. coluzzii and in semi-field experiments with
An. gambiae s.s., both members of the
An. gambiae species complex. This suggests that the attractiveness of human skin odour to these malaria-transmitting mosquitoes is not altered during administration of AL to healthy persons.
Skin odour samples collected three weeks after completion of AL-administration attracted significantly more mosquitoes than skin odour samples collected before or during administration (Fig.
3b), although mosquito choice was not influenced (Fig.
3a). This effect was only observed with
An. coluzzii in the dual-port olfactometer and not with
An. gambiae s.s. in the semi-field experiment, perhaps due to the experimental set-up or the smaller number of participants tested in the latter experiment. To ensure that increased attraction was caused by AL-administration, and not by other factors that changed over time and influenced attractiveness to mosquitoes, e.g. climatic factors, diet or physical activity, it would have been necessary to include skin odour samples from participants that had not received AL. However, other studies suggest that human skin odour and differential attractiveness of healthy humans to mosquitoes is stable over time, although very few studies have actually tested this [
33,
34]. If increased attractiveness at three weeks after AL-treatment can be confirmed in
Plasmodium-infected patients, the question is whether this is meaningful in terms of transmission. Post-treatment gametocytaemia is also found at this time point but it occurs in less than 5% of treated patients that report with gametocytes [
14], and no studies have investigated whether treated patients can infect mosquitoes at this time point. Due to this low percentage, and the relatively small increase in mosquito response to skin odour (from 52 to 66% of mosquitoes attracted), the impact of this finding on malaria transmission from humans to mosquitoes is limited at most.
Ultimately, it is essential to investigate if these findings can be translated to AL-treated
Plasmodium-infected persons.
Plasmodium-infection is known to influence human attractiveness to mosquitoes [
35‐
37] through changes in skin odour profile [
18], and gametocyte-infected red blood cells can emit mosquito-attractants [
38]. It is possible that there are interactions between ACT and
Plasmodium-infection that are not seen in healthy participants after administration of ACT.
Direct effects of AL on mosquitoes were also investigated by feeding females on human blood supplemented with AL. Results of the in vitro experiment suggest that uninfected
An. coluzzii is not affected by the drug because time until oviposition, the number of eggs in the first egg batch, and survival were the same in mosquitoes fed on control- or AL-blood (Fig.
4). Based on what is currently understood about their mode of action against
Plasmodium parasites [
23,
39,
40], it was not expected that artemether and/or lumefantrine would affect fitness of
An. coluzzii. However, differences between results in the in vitro assay and in vivo effects could arise from exposure of mosquitoes to different metabolites of artemether and lumefantrine, and different concentrations of and ratios between metabolites. In vivo, artemether and lumefantrine are quickly metabolised into dihydroartemisinin and desbutyl-lumefantrine, respectively [
24,
41]. Artemether and lumefantrine have low solubility in water and due to differences in half-life [
23], ratios between the metabolites would be different in vivo. Feeding mosquitoes on blood of ACT-treated patients could overcome these limitations. Moreover, effects may be different in mosquitoes that ingest ACT at the same time as gametocytes because there may be interactions between drugs and parasites. To verify this, it would be necessary to perform tests with
Plasmodium-infected mosquitoes. Despite these limitations, our findings suggest that mosquito fitness will not be influenced by blood-feeding on ACT-treated humans, in contrast to the endectocidal drug ivermectin that proved to have a significant impact on malaria transmission through its effect on
Anopheles mosquitoes [
42].
Conclusions
Perhaps surprisingly, there is a scarcity of studies on the effects of medication on human body odour profiles and attractiveness to mosquitoes, with the exception of a study from 1968 testing drugs and diseases as potential repellents against
Aedes mosquitoes [
43]. Here, the potential effects of AL on behaviour and fitness of malaria mosquitoes were studied. The experiments provided no evidence for a major effect of AL on human attractiveness to
An. coluzzii and
An. gambiae s.s., apart from a small increase in attractiveness 3 weeks after AL-administration. To predict whether this has any effect on post-treatment parasite transmission, it is essential to repeat our study with
Plasmodium-infected participants. Further, it is recommended that these experiments are repeated with other commonly used ACT medicines, particularly those that have limited gametocytocidal effects. For example, a recent study reported that the appearance of gametocytaemia after ACT-treatment was higher when artesunate/amodiaquine and dihydroartemisinin–piperaquine were used compared to AL [
14]. No effect of AL on fitness parameters of
An. coluzzii was found. Based on these results, it appears unlikely that AL alone has an effect on post-treatment transmission through direct or indirect effects on
Anopheles mosquitoes.
Authors’ contributions
JGdB designed the research, and analysed and interpreted the data alongside AOB, JtB and TSvD. JGdB, AOB, JtB and TSvD performed the experiments. WT contributed to interpreting the data and writing the manuscript. JGdB wrote the manuscript. All authors read and approved the final manuscript.