Anti-Malarial Drug Resistance

Malaria is a life-threatening infectious disease caused by a protozoan parasite of the genus Plasmodium. It is transmitted through the bites of infected female Anopheles mosquitoes. The global burden is estimated to be around 219 million cases in 87 countries. Natural compounds have been used primarily in the traditional medicine for thousands of years. For the treatment of malaria, natural products were used until the development of synthetic drugs, and most of the currently available anti-malarial drugs have been derived based on the compounds from these traditional medicinal plants. The current chapter tries to briefly indicate the emerging resistance against anti-malarial drugs and to discuss the recent research on natural products that have been evaluated for anti-malarial activity. Rigorous evaluation of the efficacy and safety of traditional medicines is required along with identification of active constituents in order to develop new drugs with novel mechanisms of action.

Effect of Short-Term Heating on Bioefficacy of Deltamethrin-Coated Long-Lasting Insecticidal Nets

The authors recently reported that long-lasting insecticidal nets (LLINs) distributed in Papua New Guinea (PNG) between 2013 and 2019, exhibited severely diminished efficacy to knock down and kill susceptible Anopheles mosquitoes. This coincided with a rise in malaria observed in PNG since 2015. Here, the authors show that LLIN bioefficacy is increased by heating LLINs prior to WHO cone bioassays. Unused LLINs with low bioefficacy, delivered to PNG in 2019, were heated to 120°C for 5 minutes. Cone bioassays were performed before and at 1 hour, 7 days, and 30 days after heating. This led to a significant increase in 24-hour mortality (17–61%) and 60-minute knock down (31–72%). The effect was sustained over 30 days. Bioassays are crucial in quality assurance of LLIN products. Our findings indicate that bioefficacy of LLINs can be increased by heating. This may have implications for quality assurance procedures used to assess LLINs.

Comparing Light—Emitting—Diodes Light Traps for Catching Anopheles Mosquitoes in a Forest Setting, Western Thailand

Light traps are a common method for attracting and collecting arthropods, including disease vectors such as mosquitoes. Various types of traps have been used to monitor mosquitoes in a forest in Western Thailand. In this study, four Light Emitting Diodes (LED) light sources (UV, blue, green, and red) and two fluorescent lights (white and UV) were used to trap nocturnal adult mosquitoes. These traps were used with light alone and not any additional attractant. The experiment was conducted from 18:00 to 06:00 h. on six consecutive nights, every two months, across dry, wet, and cold seasons. All specimens were first identified by morphological features and subsequently confirmed by using PCR. We collected a total of 873 specimens of 31 species in four genera, Anopheles, Aedes, Culex, and Armigeres. Anopheles harrisoni was the predominant species, followed by Aedes albopictus, Culex brevipalpis, Culex nitropunctatus, and Armigeres (Leicesteria) longipalpis. UV fluorescent light was the most effective light source for capturing forest mosquitoes, followed by UV LED, blue LED, green LED, white fluorescent, and red LED. The optimal times for collection were from 21:00 to 03:00 h in the dry season. Our results demonstrate that appropriate sampling times and light sources should be selected for optimal efficiency in vector surveillance programs.

Gene conversion explains elevated diversity in the immunity modulating APL1 gene of the malaria vector Anopheles funestus

The leucine rich repeat gene APL1 is a key component of immunity to Plasmodium and other microbial pathogens in Anopheles mosquitoes. In the malaria vector Anopheles funestus the APL1 gene has four paralogues which occur along the same chromosome arm. We show that APL1 has exceptional levels of non-synonymous polymorphism across the range of An. funestus with an average πn of 0.027 versus a genome-wide average of 0.002, and πn (and πs) is consistently high in populations across Africa. The pattern of APL1 diversity was consistent between independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide-resistance was observed with the phenotyped target-enrichment dataset. Two further innate immunity genes of the gambicin anti-microbial peptide family had πn/πs ratios greater than one, possibly driven by either positive or balancing selection. Cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, an observation discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogs, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. Gene conversion at APL1 versus alternative explanations is concordant with similarly elevated diversity in APL1 and TEP1 loci in An. gambiae. In contrast, the more closely related An. stephensi which also encodes a single-copy of APL1 does not show this elevated diversity. We hypothesise that a more open chromatin formation at the APL1 locus due to higher gene expression than its paralogues enhances gene conversion, and therefore increased polymorphism, at APL1.

Evaluating the contribution of aestivation to the persistence of malaria mosquitoes through the Sahelian dry season using stable isotopes

Data suggests the malaria vector Anopheles coluzzii persists in the Sahel by dry-season aestivation though evidence is scant. We have marked Anopheles mosquitoes using deuterium (2H) to assess the contribution of aestivation to persistence of mosquitoes through the seven-month dry season. If local aestivation is the only way A. coluzzii persists, the frequency of marked mosquitoes should remain stable throughout, whereas finding no marked mosquitoes would be evidence against aestivation. Larval sites were spiked with 2H at the end of the 2017 wet season in two Sahelian villages in Mali. We monitored 2H-enriched populations until the onset of rains. By the end of the enrichment period, 33% of A. coluzzii mosquitoes were clearly marked. Expectedly, 2H levels in marked mosquitoes degraded over time, resulting in a partial overlap of the marked and non-marked 2H distributions. We utilized three methods to estimate the fraction of marked mosquitoes in the population. Seven months after enrichment, 7% of the population had 2H values above the highest pre-enrichment value. An excess of 21% exceeded the 3rd quartile of the pre-enrichment population. A finite mixed population model showed 2.5% represented a subpopulation of marked mosquitoes with elevated 2H, compatible with our predictions. We provide evidence that aestivation is a major persistence mechanism of A. coluzzii in the Sahel, contributing at least 20% of the adults at the onset of rains, suggesting A. coluzzii utilizes multiple persistence strategies enabling its populations rapid buildup, facilitating subsequent malaria resurgence. These may complicate vector control and malaria elimination campaigns.

Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity

Plasmodium sporozoites inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver was found to be detrimental for the parasite growth, leading to the acquisition of a long-lasting immune protection. Considering IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P.berghei parasites that express murine IL-6 during liver stage development. Though IL-6 transgenic sporozoites develop into exo-erythrocytic forms in cultured hepatocytes in vitro, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6 sporozoites elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious sporozoite challenge. Collectively, this study demonstrates that parasite-encoded IL-6 impairs Plasmodium infection at the liver stage, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.

High-resolution in situ analysis of Cas9 germline transcript distributions in gene-drive Anopheles mosquitoes

Gene drives are programmable genetic elements that can spread beneficial traits into wild populations to aid in vector-borne pathogen control. Two different drives have been developed for population modification of mosquito vectors. The Reckh drive (vasa-Cas9) in Anopheles stephensi displays efficient allelic conversion through males but generates frequent drive-resistant mutant alleles when passed through females. In contrast, the AgNos-Cd1 drive (nos-Cas9) in An. gambiae achieves almost complete allelic conversion through both genders. Here, we examined the subcellular localization of RNA transcripts in the mosquito germline. In both transgenic lines, Cas9 is strictly co-expressed with endogenous genes in stem and pre-meiotic cells of the testes, where both drives display highly efficient conversion. However, we observed distinct co-localization patterns for the two drives in female reproductive tissues. These studies suggest potential determinants underlying efficient drive through the female germline. We also evaluated expression patterns of alternative germline genes for future gene-drive designs.

Impact of a Spatial Repellent (SR) product on Anopheles and non-Anopheles mosquitoes in Sumba, Indonesia

Background East Nusa Tenggara province, Indonesia, contributed to 5% of malaria cases nationally in 2020, with other mosquito-borne diseases such as dengue and filariasis also being endemic. Monitoring of spatial and temporal vector species compositions and bionomic traits is an efficient method for generating evidence towards intervention strategy optimization and meeting disease elimination goals. MethodsThe impact of a spatial repellent (SR) on human biting mosquitoes was evaluated as part of a parent cluster-randomized, double-blinded, placebo-controlled trial, in Sumba, East Nusa Tenggara. A ten-month (June 2015–March 2016) baseline study was followed by a 24-month intervention period (April 2016 to April 2018) - where half the clusters were randomly assigned either a passive transfluthrin emanator or a placebo control. ResultsHuman-landing mosquito catches documented a reduction in landing rates related to the SR. Overall, there was a 16.4% reduction (21% indoors, and 11.3% outdoors) in human biting rates (HBR) for Anopheles. For Aedes, there was a 44.3% HBR reduction indoors and a 35.6% reduction outdoors. This reduction was 38.3% indoors and 39.1% outdoors for Armigeres, and 36.0% indoors and 32.3% outdoors for Culex species. Intervention impacts on the HBRs were not significant and are attributed to large inter-household and inter cluster variation. Anopheles flavirostris, An. balabacensis and An. maculatus individually impacted the overall malaria infections hazard rate with statistically significance. Though there was SR-based protection against malaria for all Anopheles species (except An. sundaicus s.l.), only five (An. aconitus, An. kochi, An. maculatus, An. tessellatus and An. sundaicus s.l.) demonstrated statistical significance. The SR numerically reduced Anopheles parity rates indoors and outdoors when compared to the placebo. ConclusionEvidence demonstrating that Anopheles vectors bite both indoors and outdoors indicates that currently implemented indoor-based vector control tools may not be sufficient to eliminate malaria. The documented impact of the SR intervention on Aedes, Armigeres and Culex species points to its importance in combatting other vector borne diseases. Studies to determine the impact of spatial repellents on other mosquito-borne diseases is recommended.