Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi

Abstract

Background: The mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America.

Methods: To determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 μg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated.

Results: IVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions.

Conclusion: In conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

Fig 1. Schematic outline of the experiments of membrane feeding.

Four experiments were performed to determine the transmission blocking potential of IVM in either An. aquasalis or An. darlingi. Abbreviations: Ivermectin (IVM), powdered ivermectin (pIVM), metabolized ivermectin (mIVM), chloroquine (CQ), primaquine (PQ).

Fig 2. Ivermectin powdered affects Plasmodium vivax development in Anopheles aquasalis.

(A) The proportion of P. vivax infected mosquitoes is presented as a mean and standard deviation. (B) The infection intensity is presented as the number of oocysts per a single midgut (black dots) and the black lines represent the mean and standard deviation. (C) Mosquito mortality rate to 7 days post-feeding is presented as mean and standard deviation. Data from 5 independent experiments are presented in A-C. Asterisks (*) represent significant difference (P < 0.05) in relation to the control.

Fig 3. Ivermectin metabolized affects Plasmodium vivax development in Anopheles aquasalis and Anopheles darlingi.

An. aquasalis (A,C,E) and An. darlingi (B,D,F) mosquitoes were fed P. vivax with mIVM collected at various time points (0 and 4 hours; 1, 5, 10 and 14 days). (A) and (B) Proportion of P. vivax infected mosquitoes are presented as mean and standard deviation. (C) and (D) The intensity of infection is presented as the number of oocysts per a single midgut (black dots) and the black lines represent the median and standard deviation. (E) and (F) The mosquito mortality rate is presented as mean and standard deviation. Data from 5 independent experiments are presented. Asterisks (*) represent significant difference (P < 0.05) in relation to the control.

Fig 4. Activity of IVM on P. vivax infections in An. aquasalis.

P. vivax oocyst inhibition in An. aquasalis when ingesting plasma from human volunteers receiving standard single oral dosing of IVM (A) or whole blood spiked with IVM (B).

Fig 5. Ivermectin impairs Plasmodium vivax development in Anopheles aquasalis and Anopheles darlingi when administered in a double feed 4 days after infection.

An. aquasalis and An. darlingi female mosquitos were fed P. vivax blood samples. Four days after infection, two groups of mosquitos received a second uninfected blood meal with mIVM plasma from control (0 hours) or 4 hours after IVM intake. (A) The proportion of P. vivax infected An. aquasalis and (B) An. darlingi are presented as the mean and standard deviation. (C and D) The intensity of infection is presented as the number of oocysts per midgut (dots) and the black lines represent the median and standard error. Data from 5 independent experiments are presented. Asterisks (*) represent significant difference (P < 0.05) in relation to control.

Fig 6. Plasmatic ivermectin and malaria drugs have a direct effect on Plasmodium vivax infection in Anopheles aquasalis.

An. aquasalis female mosquitos were fed with unprocessed blood samples from P. vivax patients at time 0 (control) or 4 hours after different treatment regimens intake or with erythrocytes from P. vivax patients’ blood samples (before treatment) reconstituted to a hematocrit of 40% with the respective plasma after 4 hours of different treatment regimens intake. (A) The proportion of P. vivax infected mosquitos are presented as mean and standard deviation. Black bars represent the unprocessed blood and the grey bars represent the reconstitute samples. (B) The intensity of infection is presented as the number of oocysts per a single mosquito midgut (dots), black dots represent the unprocessed blood and the grey dots represent the reconstituted blood samples, and the black lines represent the median and standard error blood. Asterisks (*) represent significant difference (P < 0.05) in relation to the control.

Fig 7. Metabolized ivermectin impairs asexual P. vivax maturation.

Five P. vivax samples containing more than 80% of parasites on ring stage were evaluated. Nine different concentrations of pIVM were added to P. vivax culture. Moreover, plasma from healthy volunteers collected before (0 hours) and after 4 hours of IVM intake was added in 4 different serial dilutions (1:2) with complete medium. A drug free condition was used as the control. The number of schizonts in 200 asexual parasites was evaluated for each condition and the maturation in relation to control were determined. Asterisks (*) represents significant differences (P < 0.05) in relation to control by Kruskal-Wallis test.