Background
Artemisinin-resistant
Plasmodium falciparum isolates have been identified in the Greater Mekong Subregion (GMS) which threatens to undermine malaria elimination goals [
1]. The primary GMS
Anopheles vectors of
Plasmodium frequently feed outdoors and before people go to sleep [
2,
3], rendering classical vector control measures such as insecticide-treated bed nets and indoor residual spraying with insecticides less effective. There are currently no vector control tools in the GMS that specifically target outdoor feeding
Anopheles which is a major impediment for malaria vector control in the region. Novel vector control tools that target outdoor malaria transmission are urgently needed to support artemisinin resistance containment and malaria elimination efforts in the GMS.
Ivermectin has been shown to be lethal to over a dozen
Anopheles species worldwide [
4]. Ivermectin mass drug administration (MDA) has been suggested as a possible malaria parasite transmission control tool as it directly targets the vector at the point of human blood feeding. It is one of the few vector control measures that targets outdoor malaria transmission. Laboratory studies [
5,
6], clinical trials [
7‐
9], and field studies [
10,
11] demonstrate that ivermectin is lethal to
Anopheles gambiae at human relevant concentrations. Ivermectin MDA campaigns in Senegal, Liberia, and Burkina Faso have demonstrated that ivermectin can suppress
P. falciparum transmission by wild
An. gambiae s.l. [
11,
12]. In addition to direct mosquito-lethal effects, ivermectin suppresses development of
P. falciparum in
An. gambiae [
13,
14].
Plasmodium transmission in the GMS is complex with numerous
Anopheles species serving as primary and secondary vectors throughout the region.
Anopheles dirus s.s., found in the GMS east of Myanmar, is a primary malaria vector in forested areas and feeds predominantly outdoors on humans [
2,
15].
Anopheles minimus s.s., found throughout the GMS and parts of mainland Asia, is a primary malaria vector outside of forested areas, feeds both indoors and outdoors, and displays a variable feeding preference on humans and cattle across its range [
3,
16]. There are several secondary malaria vectors that may facilitate malaria transmission in the GMS.
Anopheles sawadwongporni, a member of the
Anopheles maculatus group, has been incriminated as a malaria vector in Thailand [
17] and
Anopheles campestris, predominantly found in rice paddies throughout the GMS is suspected to be a secondary malaria vector in Thailand [
18].
Ivermectin MDA campaigns in West Africa using a 150–200 µg/kg dose were shown to be effective at reducing
P. falciparum transmission by
An. gambiae [
11,
12]. However, not all
Anopheles may be equally susceptible to ivermectin [
4]. This implies that higher doses of ivermectin may be required during MDA to effectively target all
Anopheles in a given region. Doses of ivermectin up to 2000 µg/kg were safe and well tolerated in healthy volunteers [
19], which is ten times the amount approved for strongyloidiasis treatment in Thailand [
20]. The ivermectin dose of 800 µg/kg has been assessed in onchocerciasis-infected patients in an extended trial in Ghana [
21,
22] and repeatedly every 3 months in a trial in Cameroon [
23]. Adverse events in the ivermectin 800 µg/kg trials may be correlated with the immune response to dead microfilariae and not necessarily linked directly to ivermectin treatment. Trials in healthy and malaria infected patients without concomitant onchocerciasis infection at the ivermectin 800 μg/kg dose are warranted. The lack of adverse events in healthy volunteers at up to ivermectin doses of 2000 μg/kg [
19] would support the notion that the adverse events observed in Ghana [
21,
22] and Cameroon [
23] trials may be linked to the death and clearance of
Onchocerca volvulus parasites and not ivermectin toxicity. Several clinical trials have investigated the safety and tolerability of ivermectin at 400 and 800 µg/kg, however, to the best of our knowledge, none have assessed the pharmacokinetic properties of ivermectin at these doses, particularly in an Asian population, which is the population of interest in this study.
The mosquito-lethal effects of ivermectin on the GMS malaria vectors An. dirus s.s., An. minimus s.s., An. sawadwongporni, and An. campestris, and the sporontocidal effects of ivermectin on Plasmodium vivax in An. dirus s.s., and An. minimus s.s. were investigated. A population-based pharmacokinetic model of ivermectin and simulated concentration–time profiles after 200, 400 and 800 µg/kg doses was developed, and correlated with mosquito survivorship results to rationally select ivermectin doses for MDA use in the GMS.
Discussion
All four
Anopheles species investigated were susceptible to ivermectin at concentrations predicted to be present in humans following oral administration. This suggests that ivermectin MDA has a potential role in malaria elimination in the GMS as it is a novel vector control tool that could directly combat outdoor malaria transmission.
Anopheles dirus and
An. minimus are the two most important primary vectors of malaria in the GMS while
An. campestris and
An. sawadwongporni are potential secondary vectors [
31]. Since
An. dirus is arguably the most important GMS malaria vector it is critical that ivermectin MDA deliver a dose of ivermectin high enough to control this vector. Population peak ivermectin concentrations after a single oral dose of 200 µg/kg did not reach the 7-day-LC
50 of
An. dirus. However, pharmacokinetic modelling and simulation presented here suggest that a single oral ivermectin dose of 400 or 800 µg/kg results in concentrations that surpasses the ivermectin 7-day-LC
50 for
An. dirus (Table
3). Based on the
An. dirus in vitro survivorship results, pharmacokinetic model output and extensive safety data discussed below, the ivermectin 400 µg/kg dose appears to be the ideal minimal dose to administer during MDA for malaria control in the GMS.
The simulated time above the
An. dirus ivermectin 7-day-LC
50 at ivermectin doses of 400 µg/kg (0.4 days) or 800 µg/kg (1.1 days) would not seem to deliver a substantial mosquito-killing window following ivermectin MDA. However, it should be noted that the 200 µg/kg dose would only provide a simulated 0.6 days above the
An. gambiae 5-day-LC
50 value of 22.4 ng/ml [
5], and yet ivermectin MDAs in Senegal at doses of 150 µg/kg substantially reduced
An. gambiae 5-day survivorship by 43.6% for up to 6 days post MDA [
10]. Furthermore, the pharmacokinetic model predicts that only 0.432 ng/ml of ivermectin parent compound would be present in a typical patient of 55 kg body weight 6 days post MDA at the 150 µg/kg dose, which is well below the concentration capable of killing
An. gambiae. This clearly illustrates that in vitro mosquito membrane feeds and pharmacokinetic predictions of parent compound likely underestimate the full mosquito-lethal potential of ivermectin-treated humans. One possible explanation may be that ivermectin produces active mosquito-lethal in vivo metabolites with different pharmacokinetic properties that extend the duration of mosquito-lethal effect beyond that of the parent compound. Human liver microsomes have been used to characterize some of the ivermectin metabolites [
32]. A small (n = 4) mass balance study in humans determined that mean peak plasma concentration of metabolites was 2.5-fold greater than that of the parent compound and the effective half-lives of the metabolites was approximately 2.9 days while the parent compound half-life was 11.8 h [
33]. Further attention to the characterization of ivermectin mosquito-lethal metabolites is warranted, especially in light of the novel long-lasting ivermectin formulations in development for human use [
34‐
36].
The final population pharmacokinetic model developed in this study described ivermectin concentrations in healthy volunteers satisfactorily. Overall parameter estimates were similar to that reported previously in healthy volunteers and patients with onchocerciasis [
37,
38]. Simulation-based diagnostics demonstrated a good predictive performance, which suggests that the developed model is suitable to use for simulations. However, the model was developed on data from a single oral dose of 200 µg/kg and extrapolations beyond that (i.e. simulations of 400 and 800 µg/kg doses) assumes that ivermectin shows dose linearity at this dosing range [
19]. Body weight, implemented as allometric function, produces a biologically plausible covariate relationship [
39,
40] between ivermectin exposure and body weight so that the developed pharmacokinetic model can be used to simulate other populations at risk, such as children. However, the pharmacokinetic properties of a drug can be very different in children and adults due to the rapid change in body size, organ function, body composition, and enzyme maturation, which occur during the early years of life. Prospective clinical trials are urgently needed in children since there are currently no pharmacokinetic assessments of ivermectin in children and adolescents below 17 years of age. A previous study [
8] reported that female participants and participants with higher body mass index (BMI) had higher day-7 ivermectin concentrations. However, BMI was not found to be a significant covariate on any pharmacokinetic parameters in the current analysis, perhaps due to the relatively narrow range of BMI (17.8–22.8 kg/m
2) studied here. Simulation of ivermectin concentration–time profiles at different dosing regimens were also performed in a previous study [
41]. The authors obtained pharmacokinetic parameters from the literature, based on American healthy volunteers and assuming a 30% inter-individual variability in each parameter. Their simulated concentration–time profile after a single dose of 800 µg/kg of ivermectin resulted in an average peak concentration of 108.1 ng/ml [
41], which is somewhat lower than the value simulated using the model developed here (174 ng/ml).
The ivermectin dose of 400 µg/kg has been investigated in thousands of adults and children, in healthy and infected persons (e.g. lymphatic filariasis, onchocerciasis, loaisis, ascariasis, trichuriasis, hookworm and lice), in more than 20 clinical trials in ten countries, including India [
42], Cameroon [
23], Ghana [
43], Gabon [
44], Sri Lanka [
45‐
47], Mali [
48], Papua New Guinea [
49,
50], French Polynesia [
51‐
58], Brazil [
59‐
62], Haiti [
63,
64], and France [
65]. Repeated ivermectin administration at doses of 400 µg/kg was safe in two trials in adults who were treated every two weeks for 12 weeks in Sri Lanka [
45] and Brazil [
61]. The ivermectin dose of 400 µg/kg was deemed safe enough to perform several rounds of MDA to thousands of people in India [
42], Cameroon [
23], Papua New Guinea [
49], and French Polynesia [
56,
57] with minimal adverse events reported. Ramaiah and colleagues led the largest MDA trial of ivermectin 400 µg/kg study to date, wherein five entire villages, roughly 10,000 people, including children and adults of both gender, were treated by MDA nine times over an 11-year period. The 400 µg/kg ivermectin dose is now recommended for lymphatic filariasis when twice yearly ivermectin 200 µg/kg MDA cannot logistically be performed [
66]. This extensive safety data should justify the use of ivermectin 400 µg/kg for MDA in the GMS.
The sporogony experiments conducted here clearly indicate a sporontocidal effect of ivermectin against
P. vivax in both
An. dirus and
An. minimus (Figs.
5,
6). Both
P. vivax oocyst prevalence and intensity were significantly reduced when ivermectin compound at the LC
25 and LC
5 when ingested concomitantly (DPI 0) with parasites in
An. dirus and
An. minimus. A previous study found that ivermectin compound significantly reduced
P. falciparum prevalence in
An. gambiae when ingested concomitantly (DPI 0) at the ivermectin LC
25 but not the LC
5, and had no effect on oocyst intensity at either concentration [
13]. A sporontocidal effect would reduce onward transmission from infected persons that received ivermectin MDA while gametocytaemic. This may be more relevant for transmission suppression for
P. vivax than
P. falciparum as
P. vivax gametocytes mature much more rapidly and are therefore present before people become ill enough to seek treatment [
67]. These studies only assess the sporontocidal effect of ivermectin in the mosquito, future studies should investigate the potential gametocytocidal action of ivermectin.
There was no effect of ivermectin LC
5 compound on
P. vivax oocyst prevalence (Fig.
7) or intensity (Additional file
1: Figure S2) when ingested by
An. dirus at DPI −3. This is in direct contrast to previous findings which showed ivermectin compound LC
5 at DPI −3 reduced oocyst prevalence of
P. falciparum in
An. gambiae [
13]. Ingestion of blood from ivermectin-treated cattle 4 days before ingestion of field isolates of
P. falciparum from infected patients did not have a sporontocidal effect [
68]. Ivermectin LC
25 compound failed to reduce
P. vivax sporozoite prevalence in
An. dirus when ingested at DPI 6 or 9 (Fig.
7), while there was a significant sporontocidal effect at the ivermectin LC
25 at DPI 6 and 9 for
P. falciparum in
An. gambiae [
13]. This indicates that ivermectin can have differential sporontocidal impact with different
Anopheles and
Plasmodium species combinations.
Previously it was suggested that ivermectin sporontocidal effect may be due to direct effects on the mosquito, specifically ivermectin delaying or altering formation of the peritrophic matrix [
13]. Indeed, ivermectin has been shown to delay and alter peritrophic matrix formation in
Aedes aegypti [
69], delay blood meal digestion in
An. gambiae [
5], and upregulates peritrophic matrix gene expression following ivermectin ingestion in
An. gambiae [
70]. However, limited investigations by two other laboratories were unable to identify a sporontocidal effect of ivermectin against
P. falciparum NF54 strain in
An. gambiae or
Anopheles stephensi [
8] or with field isolates of
P. falciparum in
An. gambiae [
71]. This suggests more complex interaction of ivermectin on mosquito and parasite interaction, possibly by influencing mosquito midgut microbiota. The mosquito midgut microbiota composition has recently been shown to dramatically alter
Anopheles immune response and thus
Plasmodium infection [
72]. Original investigations of avermectin, the biological precursor of ivermectin, suggested no direct effect on a range of bacteria species [
73]. However, more recent evidence indicates that avermectin can inhibit growth of
Staphylococcus aureus [
74], and ivermectin can inhibit growth of
Chlamydia trachomatis [
75] and
Mycobacterium tuberculosis [
76]. This recent antibacterial evidence suggests that ivermectin may influence mosquito midgut microbiota composition, which could in turn alter
Plasmodium infection. Recently, the midgut microbiota composition was shown to significantly alter formation of the peritrophic matrix [
77], thus possible alteration of the midgut microbiota by ivermectin may influence formation of the peritrophic matrix and in turn alter
Plasmodium infection. Since different insectaries around the world likely have different midgut bacteria microbiota compositions in colonized mosquitoes, this may explain the differences observed by different laboratories when investigating ivermectin sporontocidal effects. Much remains to be explored to determine the sporontocidal mode of action of ivermectin.
Concomitant ingestion of
P. vivax and ivermectin LC
25 failed to alter
An. dirus survivorship compared to ivermectin LC
25 alone (Fig.
8). This in contrast to the finding that
P. falciparum-infectious
An. gambiae that ingested ivermectin LC
25 at DPI 14 were significantly, albeit modestly, more susceptible to ivermectin compared to uninfected
An. gambiae [
13]. There were some caveats to the
P. vivax and
An. dirus survival study in that mosquitoes had to be transported between and housed in two different insectaries and
P. vivax blood was collected freshly from a single donor in sodium heparin tubes and uninfected blood from multiple donors was stored briefly in CPDA-1 bags. There was no significant difference in
An. dirus survivorship when fed a control or ivermectin LC
25 meal mixed with blood collected in CPDA-1 or sodium heparin tubes (Additional file
1: Figure S3, additional information text). Future investigations are warranted to determine whether
Plasmodium infection in
Anopheles alters susceptibility to ivermectin.
During ivermectin MDAs for malaria, numerous neglected tropical diseases (NTDs) in the GMS would be affected, including lymphatic filariasis, scabies, lice, gnathostomiasis, and soil-transmitted helminths (STHs) such as strongyloidiasis, ascariasis and trichuriasis. It has been estimated that 50% of persons in resource-poor communities in the GMS have one or more STH [
78], and strongyloidiasis afflicts between 40 to 60% of persons in rural Cambodia [
79] and Laos [
80]. Ivermectin was found to be very effective at treating strongyloidiasis and repeated MDAs would further benefit afflicted communities as re-infection rates in Cambodia can be quite high [
81]. Years of experience with ivermectin MDA for onchocerciasis in Africa have demonstrated that treated persons clearly recognize and appreciate the secondary benefits that ivermectin treatment has on NTDs [
82].
MDA with artemisinin combination therapy (ACT) is being piloted in the GMS [
1] and Africa [
83]. One problem facing ACT MDA is that asymptomatic persons may not perceive a direct personal benefit of clearing malaria parasites. However, if ivermectin were incorporated into ACT MDA then this may improve compliance as persons could observe a direct personal benefit to MDA participation by reducing NTD burdens. Ivermectin and ACT MDA for malaria control, when rationally deployed in targeted hotspot areas of active
Plasmodium transmission, would act in concert by clearing infected persons of their malaria parasites while reducing the transmission potential of the extant mosquito population. This could essentially reset malaria transmission when the next wave of naïve
Anopheles emerge from the larval habitat and feed on a population cleared of their malaria parasites. Modelling suggests that if ivermectin is added to anti-malarial drug MDA this will reduce the number of MDAs and time required to achieve elimination [
84]. The combination of ivermectin and artemether-lumefantrine was shown to be very safe [
8]. Two clinical trials are currently being conducted to assess the safety, tolerability, pharmacokinetics and mosquito-lethal efficacy of ivermectin and dihydroartemisinin-piperaquine in Thailand (NCT02568098) and Kenya (NCT02511353). If ivermectin can integrate with ACT MDA, then this could become a powerful new tool for malaria elimination.