Emergence Of Parasites Research Articles

Advancements and Challenges in Developing Malaria Vaccines: Targeting Multiple Stages of the Parasite Life Cycle.

Malaria, caused by Plasmodium species, remains a major global health concern, causing millions of deaths annually. While the introduction of the RTS,S vaccine has shown promise, there is a pressing need for more effective vaccines due to the emergence of drug-resistant parasites and insecticide-resistant vectors. However, the complex life cycle and genetic diversity of the parasite, technical obstacles, limited funding, and the impact of the 2019 pandemic have hindered progress in malaria vaccine development. This review focuses on advancements in malaria vaccine development, particularly the ongoing clinical trials targeting antigens from different stages of the Plasmodium life cycle. Additionally, we discuss the rationale, strategies, and challenges associated with vaccine design, aiming to enhance the immune response and protective efficacy of vaccine candidates. A cost-effective and multistage vaccine could hold the key to controlling and eradicating malaria.

Lumefantrine pressure selects nonsynonymous mutation in cysteine desulfurase IscS gene in the rodent malaria parasite Plasmodium berghei ANKA

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ) are the essential long-acting partner drugs in the artemisinin-based combination therapies (ACTs) treatment regimens globally. The recent report on the emergence of artemisinin-resistant parasites portends an imminent failure of the partner drug in clearing the high residual parasite densities. Understanding the resistance mechanisms to partner drugs remains critical for tracking resistant parasites. Cysteine desulfurase IscS (nfs1), one of the proteins involved in the iron-sulfur (FeS) biogenesis pathway, has been implicated in mediating malaria parasite drug resistance. Methods: Using the rodent malaria parasites Plasmodium berghei ANKA in mice, we assessed whether the nfs1 gene is associated with LM, PQ, and AQ resistance. We first verified the stability of the LM, PQ, and AQ-resistant parasites in the standard 4-Day Suppressive Test. By means of PCR and sequencing analysis, we probed for single nucleotide polymorphisms (SNPs) in the nfs1 gene. Using qPCR, we then measured the expression of the nfs1 gene in resistant parasites relative to the drug-sensitive parent parasites. Results: Our analyses of nfs1 reveal a non-synonymous Gln142Arg mutation in the LM and PQ-resistant parasites. This mutation was not detected in the AQ-resistant parasites. The mRNA quantification of the nfs1 gene reveals differential expression in both LM and PQ-resistant parasites. Conversely, nfs1 expression remained unchanged in the AQ-resistant parasites. Conclusion: Our data suggest that LM and PQ selection pressure induces nonsynonymous mutation and differential expression of the nfs1 gene in Plasmodium berghei. Collectively, these findings provide a premise for investigating LM and PQ resistance mechanisms in both P. berghei and P. falciparum.

Formulation and optimization of ivermectin nanocrystals for enhanced topical delivery

The increasing resistance to antiparasitic drugs and limited availability of new agents highlight the need to improve the efficacy of existing treatments. Ivermectin (IVM) is commonly used for parasite treatment in humans and animals, however its efficacy is not optimal and the emergence of IVM-resistant parasites presents a challenge. In this context, the physico-chemical characteristics of IVM were modified by nanocrystallization to improve its equilibrium water-solubility and skin penetration, potentially improving its therapeutic effectiveness when applied topically. IVM-nanocrystals (IVM-NC) were prepared using microfluidization technique. The impact of several process/formulation variables on IVM-NC characteristics were studied using D-optimal statistical design. The optimized formulation was further lyophilized and evaluated using several in vitro and ex vivo tests. The optimal IVM-NC produced monodisperse particles with average diameter of 186 nm and polydispersity index of 0.4. In vitro results showed an impressive 730-fold increase in the equilibrium solubility and substantial 24-fold increase in dissolution rate. Ex vivo permeation study using pig's ear skin demonstrated 3-fold increase in dermal deposition of IVM-NC. Additionally, lyophilized IVM-NC was integrated into topical cream, and the resulting drug release profile was superior compared to that of the marketed product. Overall, IVM-NC presents a promising approach to improving the effectiveness of topically applied IVM in treating local parasitic infections.

Emergence of putative energy parasites within Clostridia revealed by genome analysis of a novel endosymbiotic clade.

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0-1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.

Tackling the emerging Artemisinin-resistant malaria parasite by modulation of defensive oxido-reductive mechanism via nitrofurantoin repurposing

Oxidative stress-mediated cell death has remained the prime parasiticidal mechanism of front line antimalarial, artemisinin (ART). The emergence of resistant Plasmodium parasites characterized by oxidative stress management due to impaired activation of ART and enhanced reactive oxygen species (ROS) detoxification has decreased its clinical efficacy. This gap can be filled by development of alternative chemotherapeutic agents to combat resistance defense mechanism. Interestingly, repositioning of clinically approved drugs presents an emerging approach for expediting antimalarial drug development and circumventing resistance. Herein, we evaluated the antimalarial potential of nitrofurantoin (NTF), a clinically used antibacterial drug, against intra-erythrocytic stages of ART-sensitive (Pf3D7) and resistant (PfKelch13R539T) strains of P. falciparum, alone and in combination with ART. NTF exhibited growth inhibitory effect at submicro-molar concentration by arresting parasite growth at trophozoite stage. It also inhibited the survival of resistant parasites as revealed by ring survival assay. Concomitantly, in vitro combination assay revealed synergistic association of NTF with ART. NTF was found to enhance the reactive oxygen and nitrogen species, and induced mitochondrial membrane depolarization in parasite. Furthermore, we found that exposure of parasites to NTF disrupted redox balance by impeding Glutathione Reductase activity, which manifests in enhanced oxidative stress, inducing parasite death. In vivo administration of NTF, alone and in combination with ART, in P. berghei ANKA-infected mice blocked parasite multiplication and enhanced mean survival time. Overall, our results indicate NTF as a promising repurposable drug with therapeutic potential against ART-sensitive as well as resistant parasites.

First molecular data on the human roundworm Ascaris lumbricoides species complex from the Bronze and Iron Age in Hallstatt, Austria

Palaeoparasitological studies can provide valuable information on the emergence, distribution, and elimination of parasites during a particular time in the past. In the prehistoric salt mines of Hallstatt, located in the Austrian Alps, human faeces have been conserved in salt. The aim of this study was to recover ancient DNA of intestinal parasites from these coprolites. Altogether, 35 coprolites from the Hallstatt salt mines, dating back to the Bronze Age mining phase (1158–1063 BCE) and the Iron Age mining phase (750–662 BCE), respectively, were analysed by microscopy and molecular methods. In 91% of the coprolite samples, eggs of soil-transmitted helminths (STH), namely of Trichuris and/or Ascaris were detected by light microscopy. The Ascaris eggs were exceptionally well preserved. For further analysis, DNA was extracted from the palaeofaecal samples and species-specific primers targeting different genes were designed. While amplification of Trichuris DNA remained unsuccessful, sequence data of A. lumbricoides species complex were successfully obtained from 16 coprolites from three different genes, the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), the mitochondrial cytochrome B gene (cytB) and the mitochondrial NADH dehydrogenase subunit 1 gene (nadh1). Importantly, these included two Ascaris sequences from a coprolite from the Bronze Age, which to the best of our knowledge are the first molecular data of this genus from this period.

Distinct evolution of type I glutamine synthetase in Plasmodium and its species-specific requirement

Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids. Interestingly, a putative gene for glutamine synthetase (GS) is retained despite glutamine being an abundant amino acid in human and mosquito hosts. Here we show Plasmodium GS has evolved as a unique type I enzyme with distinct structural and regulatory properties to adapt to the asexual niche. Methionine sulfoximine (MSO) and phosphinothricin (PPT) inhibit parasite GS activity. GS is localized to the parasite cytosol and abundantly expressed in all the life cycle stages. Parasite GS displays species-specific requirement in Plasmodium falciparum (Pf) having asparagine-rich proteome. Targeting PfGS affects asparagine levels and inhibits protein synthesis through eIF2α phosphorylation leading to parasite death. Exposure of artemisinin-resistant Pf parasites to MSO and PPT inhibits the emergence of viable parasites upon artemisinin treatment.

MMV006087 is a potent Malaria Box compound against Plasmodium falciparum clinical parasites

The emergence of drug-resistant malaria parasites to artemisinin and its partner drugs highlights the need to increase the arsenal of new antimalarials with novel mechanisms of action. To help achieve this aim, this study tested the potency of three Malaria Box compounds (MMV006087, MMV085203, and MMV008956) against five laboratory strains and twenty clinical isolates of Plasmodium falciparum using optimized in vitro growth inhibitory assays. The results were compared to the response from four standard antimalarials-artesunate, chloroquine, mefloquine, and halofantrine. From the results, MMV006087 was the most potent compound with an average IC50 of 22.13 nM compared to MMV085203 (average IC50 of 137.90 nM) and MMV008956 (average IC50 of 262.30 nM). On average, the laboratory strains were also less susceptible to the three Malaria Box compounds (average IC50 of 162.30 nM) compared to the clinical isolates (average IC50 of 135.40 nM). Additionally, MMV006087 was less potent than artesunate but twice more efficacious than chloroquine against the laboratory strains and clinical isolates. The data from this study validate the potency of MMV006087 and MMV085203 as promising antimalarials worthy of further exploration. This study further substantiates the need to include clinical isolates in antimalarial compound screening activities.

Role of Nanomedicines in Controlling Malaria: A review.

Malaria has created havoc since time immemorial. It has actually become a major health concern due to its high prevalence in developing countries where poor sanitary conditions facilitate the seasonal breeding of the vector, the female Anopheles mosquito. Even after tremendous advancements in pest control and pharmacology science, managing this disease has not been successful, and the cure for this deadly infection has not proven effective lately. The various conventional drugs used are chloroquine, primaquine, mefloquine, atovaquone, quinine, artemisinin etc. All of these have one or other major disadvantages like multi-drug resistance, high dose requirements, aggravated toxicity, non-specificity of conventional drugs, and the emergence of drug-resistant parasites. Therefore, it is necessary to surpass these limitations and look for an alternative to curb the spread of this disease using an emerging technology platform. Nanomedicine is showing promise as an effective alternative tool for the management of malaria. The idea of this tool resonates well with David J. Triggle's outstanding suggestion "The chemist is as the astronaut, searching for biologically useful space in the chemical universe. This review presents a detailed discussion on various nanocarriers, their mode of action and future perspective in treating malaria. Nanotechnology-based drug delivery methods are highly specific, require a lower dose, offer more bioavailability with prolonged drug release and stay in the body longer. Recent nano drug encapsulation and delivery vehicles comprise nanocarriers like liposomes, and organic and inorganic nanoparticles, emerging as promising alternatives for malaria management.

Ecological Interdependence of Pollution, Fish Parasites, and Fish in Freshwater Ecosystems of Turkey

Records of mass fish deaths were found in different data sources for this study. A map of mass fish deaths in Turkey was also realized for the first time. We aimed to present a review of the distribution of mass fish deaths in the freshwater ecosystems of Turkey, mostly present in the Marmara Region and Aegean Sea Region, where there are intensive industrial and agricultural activities. Fish parasites generally occur in equilibrium with their hosts in natural environments. In the freshwater ecosystems of Turkey, which are highly affected by human activities and have extremely rich natural ichthyofauna, the negative effects of pollution on environmental parameters, which can directly affect the fish, and the emergence and overextension of fish parasites can cause a supplementary synergic direct negative effect transposition in mass fish deaths due to the deterioration of host immunity and to the wounds induced in fish where bacterial, viral, and fungal pathogens can be effective. Finally, these factors can influence the fish rate of survival and skew the structure of fish populations. Mass fish deaths have been frequently reported in Turkey, but are usually only explained by pollution as a single accepted anthropogenic stressor. Together with pollution, a supplementary induced bioecological stressor, the qualitative and quantitative characteristics of the fish parasites’ association variations should be assessed and monitored as potential complex precursor indicators of fish communities’ structural degradation and freshwater ecosystems’ dreadful conditions. Fish parasites as biomonitor species should be used to identify the effects of pollution in Turkish ecosystems, and not only in freshwater ecosystems, in the future. An optimum management plan for freshwater ecosystems should include all the physico-chemical factors, fish parasites, and fish elements involved in permanent assessment and monitoring activities.

Functional validation of the truncated UNC-63 acetylcholine receptor subunit in levamisole resistance

Levamisole is a broad-spectrum anthelmintic which permanently activates cholinergic receptors from nematodes, inducing a spastic paralysis of the worms. Whereas this molecule is widely used to control parasitic nematodes impacting livestock, its efficacy is compromised by the emergence of drug-resistant parasites. In that respect, there is an urgent need to identify and validate molecular markers associated with resistance. Previous transcriptomic analyses revealed truncated cholinergic receptor subunits as potential levamisole resistance markers in the trichostrongylid nematodes Haemonchus contortus, Telodorsagia circumcincta and Trichostrongylus colubriformis. In the present study we used the Xenopus oocyte, as well as the free-living model nematode Caenorhabditis elegans, as heterologous expression systems to functionally investigate truncated isoforms of the levamisole-sensitive acetylcholine receptor (L-AChR) UNC-63 subunit. In the Xenopus oocyte, we report that truncated UNC-63 from C. elegans has a strong dominant negative effect on the expression of the recombinant C. elegans L-AChRs. In addition, we show that when expressed in C. elegans muscle cells, truncated UNC-63 induces a drastic reduction in levamisole susceptibility in transgenic worms, thus providing the first known functional validation for this molecular marker in vivo.

Emergence of Ancylostoma caninum parasites with the benzimidazole resistance F167Y polymorphism in the US dog population

BackgroundAnthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the β-tubulin isotype-1 gene and to determine the geographic distribution. MethodsAllele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum β-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by β-tubulin gene sequencing and allele frequencies were determined. ResultsThe Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by β-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%. ConclusionsThis data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.

Lumefantrine pressure selects nonsynonymous mutation in cysteine desulfurase IscS gene in the rodent malaria parasite Plasmodium berghei ANKA

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ) are the essential long-acting partner drugs in the artemisinin-based combination therapies (ACTs) treatment regimens globally. The recent report on the emergence of artemisinin-resistant parasites portends an imminent failure of the partner drug in clearing the high residual parasite densities. Understanding the resistance mechanisms to partner drugs remains critical for tracking resistant parasites. Cysteine desulfurase IscS (nfs1), one of the proteins involved in the iron-sulfur (FeS) biogenesis pathway, has been implicated in mediating malaria parasite drug resistance. Methods: Using the rodent malaria parasites Plasmodium berghei ANKA in mice, we assessed whether the nfs1 gene is associated with LM, PQ, and AQ resistance. We first verified the stability of the LM, PQ, and AQ-resistant parasites in the standard 4-Day Suppressive Test. By means of PCR and sequencing analysis, we probed for single nucleotide polymorphisms (SNPs) in the nfs1 gene. Using qPCR, we then measured the expression of the nfs1 gene in resistant parasites relative to the drug-sensitive parent parasites. Results: Our analyses of nfs1 reveal a non-synonymous Gln142Arg mutation in the LM and PQ-resistant parasites. This mutation was not detected in the AQ-resistant parasites. The mRNA quantification of the nfs1 gene reveals differential expression in both LM and PQ-resistant parasites. Conversely, nfs1 expression remained unchanged in the AQ-resistant parasites. Conclusion: Our data suggest that LM and PQ selection pressure induces nonsynonymous mutation and differential expression of the nfs1 gene in Plasmodium berghei. Collectively, these findings provide a premise for investigating LM and PQ resistance mechanisms in both P. berghei and P. falciparum.

A snapshot of the prevalence of dihydropteroate synthase-431V mutation and other sulfadoxine-pyrimethamine resistance markers in Plasmodium falciparum isolates in Nigeria

BackgroundMalaria is a major public health issue with substantial risks among vulnerable populations. Currently, the World Health Organization (WHO) recommends SP-IPTp in the second and third trimesters. However, the efficacy of SP-IPTp is threatened by the emergence of sulfadoxine-pyrimethamine resistant malaria parasites due to single nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. This study aimed to assess the current prevalence of Pfdhfr/Pfdhps mutations in P. falciparum isolates collected from individuals residing in Ile-Ife, Nigeria, and also present maps of the prevalence of Pfdhps 431V and 581G within Nigeria and surrounding countries.MethodsBetween October 2020 and April 2021, samples were collected as dried blood spots among 188 participants who showed malaria positivity with a histidine-rich-protein-based rapid diagnostic test (RDT). Nested PCR assays were used to confirm falciparum in the samples with RDT positivity, and to amplify fragments of the Pfdhfr/Pfdhps genes followed by targeted amplicon sequencing. Published data since 2007 on the prevalence of the Pfdhps genotypes in Nigeria and the neighbouring countries were used to produce maps to show the distribution of the mutant genotypes.ResultsOnly 74 and 61 samples were successfully amplified for the Pfdhfr and Pfdhps genes, respectively. At codons resulting in N51I, C59R, and S108N, Pfdhfr carried mutant alleles of 97.3% (72/74), 97.3% (72/74) and 98.6% (73/74), respectively. The Pfdhps gene carried mutations at codons resulting in amino acid changes at 431–436-437–540-581–613; I431V [45.9%, (28/61)], A581G [31.1% (19/61)] and A613S [49.2% (30/61)]. Constructed haplotypes were mainly the triple Pfdhfr mutant 51I-59R-108N (95.9%), and the most common haplotypes observed for the Pfdhps gene were the ISGKAA (32.8%), ISGKGS (8.2%), VAGKAA (14.8%), VAGKAS (9.8%) and VAGKGS (14.8%). In the context of the previously published data, a high prevalence of 431V/581G mutations was found in the study population. It seems quite evident that the Pfdhps 431V, 581G and 613S often co-occur as Pfdhps-VAGKGS haplotype.ConclusionThis study showed that the prevalence of VAGKGS haplotype seems to be increasing in prevalence. If this is similar in effect to the emergence of 581G in East Africa, the efficacy of SP-IPTp in the presence of these novel Pfdhps mutants should be re-assessed.

Ethnobotanical survey of the traditional antiparasitic use of medicinal plants in humans and animals in Laghouat (Southern Algeria).

An ethnobotanical survey was carried out among the inhabitants of the Aflou region of Laghouat (Southern Algeria). This study was considered as a first step toward the identification of new bioactive antiparasitic molecules. The preservation and documentation of this traditional knowledge will ensure its continuity and transmission from one generation to another, especially because of the emergence of resistant parasites and the lack of references caused by the lack of work in this area; therefore, we intended to inventory and collect the maximum amount of information on medicinal plants that are traditionally used by the local population as antiparasitic in humans and animals (small ruminants, cattle, and livestock). The information was collected using open interviews; the ethnobotanical survey was carried out in the area mentioned above from April to July 2021 using a semi-structured questionnaire and a global sample of 200 respondents. The data were analyzed using the System Package for the Social Sciences software and Microsoft Excel 2010 using the following quantitative indices: Relative frequency of citation (RFC), family importance value (FIV), fidelity level, and informant consensus factor (ICF). The investigation uncovered the antiparasitic use of 58 plant species belonging to 30 families. The family Asteraceae had the highest FIV (FIV = 0.23). The pathology with the highest degree of agreement among the informants was genitourinary parasitosis (ICF = 0.930). The species that was most commonly cited by the local population was Artemisia herba-alba Asso (RFC = 1), and the foliage was the most commonly used part (46.4%). Infusion (38.8%) was the most-used preparation for remedies. This investigation revealed a rich ethnopharmacological knowledge in southern Algeria; therefore, the data gathered in this survey may be utilized to create novel antiparasitic compounds with activity in humans and animals.

Antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, on Leishmania (Leishmania) amazonensis.

Leishmaniasis, a neglected disease caused by the parasite Leishmania, is treated with drugs associated with high toxicity and limited efficacy, in addition to constant reports of the emergence of resistant parasites. In this context, snake serums emerge as good candidates since they are natural sources with the potential to yield novel drugs. We aimed to show the antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, against Leishmania (Leishmania) amazonensis. Promastigotes forms were exposed to γCdcPLI, and we assessed the parasite viability and cell cycle, as well as invasion and proliferation assays. Despite the low cytotoxicity effect on macrophages, our data indicate that γCdcPLI has a direct effect on parasites promoting an arrest in the G1 phase and reduction in the G2/M phase at the highest dose tested. Moreover, this PLA2 inhibitor reduced the parasite infectivity when promastigotes were pre-treated. Also, we demonstrated that the γCdcPLI treatment modulated the host cell environment impairing early and late steps of the parasitism. γCdcPLI is an interesting tool for the discovery of new essential targets on the parasite, as well as an alternative compound to improve the effectiveness of the leishmaniasis treatment.

Ring-stage growth arrest: Metabolic basis of artemisinin tolerance in Plasmodium falciparum

The emergence and spread of artemisinin-tolerant malaria parasites threatens malaria control programmes worldwide. Mutations in the propeller domain of the Kelch13 protein confer Plasmodium falciparum artemisinin resistance (ART-R). ART-R is linked to the reduced susceptibility of temporary growth-arrested ring-stage parasites, but the metabolic mechanisms remain elusive. We generated two PfKelch13 mutant lines via CRISPR-Cas9 gene editing which displayed a reduced susceptibility accompanied by an extended ring stage. The metabolome of ART-induced ring-stage growth arrest parasites carrying PfKelch13 mutations showed significant alterations in the tricarboxylic acid (TCA) cycle, glycolysis, and amino acids metabolism, pointing to altered energy and porphyrin metabolism with metabolic plasticity. The critical role of these pathways was further confirmed by altering metabolic flow or through chemical inhibition. Our findings uncover that the growth arrestment associated with ART-R is potentially attributed to the adaptative metabolic plasticity, indicating that the defined metabolic remodeling turns out to be the trigger for ART-R.

Population genomics reveal distinct and diverging populations of An. minimus in Cambodia

Anopheles minimus is an important malaria vector throughout its wide geographic range across Southeast Asia. Genome sequencing could provide important insights into the unique malaria transmission dynamics in this region, where many vector species feed and rest outdoors. We describe results from a study using Illumina deep whole-genome sequencing of 302 wild-caught An. minimus collected from three Cambodian provinces over several years (2010, 2014, 2016) and seasons to examine the level of population structure and genetic diversity within this species. These specimens cluster into four distinct populations of An. minimus s.s., with two populations overlapping geographically. We describe the underlying genetic diversity and divergence of these populations and investigated the genetic variation in genes known to be involved in insecticide resistance. We found strong signals of selection within these An. minimus populations, most of which were present in the two Northeastern Cambodian populations and differ from those previously described in African malaria vectors. Cambodia is the focus of the emergence and spread of drug-resistant malaria parasites, so understanding the underlying genetic diversity and resilience of the vectors of these parasites is key to implementing effective malaria control and elimination strategies. These data are publicly available as part of the MalariaGEN Vector Observatory, an open access resource of genome sequence data.

Plasmodium cynomolgi in humans: current knowledge and future directions of an emerging zoonotic malaria parasite.

Plasmodium cynomolgi (Pcy), a simian malaria parasite, is a recent perfect example of emerging zoonotic transfer in human. This review summarizes the current knowledge on the epidemiology of natural Pcy infections in humans, mosquitoes and monkeys, along with its biological, clinical and drug sensitivity patterns. Knowledge gaps and further studies on Pcy in humans are also discussed. This parasite currently seems to be geographically limited in South-East Asia (SEA) with a global prevalence in human ranging from 0 to 1.4%. The Pcy infections were reported in local SEA populations and European travelers, and range from asymptomatic carriage to mild/moderate attacks with no evidence of pathognomonic clinical and laboratory patterns but with Pcy strain-shaped clinical differences. Geographical distribution and competence of suitable mosquito vectors and non-primate hosts, globalization, climate change, and increased intrusion of humans into the habitat of monkeys are key determinants to emergence of Pcy parasites in humans, along with its expansion outside SEA. Sensitization/information campaigns coupled with training and assessment sessions of microscopists and clinicians on Pcy are greatly needed to improve data on the epidemiology and management of human Pcy infection. There is a need for development of sensitive and specific molecular tools for individual diagnosis and epidemiological studies. The development of safe and efficient anti-hypnozoite drugs is the main therapeutic challenge for controlling human relapsing malaria parasites. Experience gained from P. knowlesi malaria, development of integrated measures and strategies-ideally with components related to human, monkeys, mosquito vectors, and environment-could be very helpful to prevent emergence of Pcy malaria in humans through disruption of transmission chain from monkeys to humans and ultimately contain its expansion in SEA and potential outbreaks in a context of malaria elimination.

A comprehensive overview of the existing microbial symbionts in mosquito vectors: An important tool for impairing pathogen transmission

The emergence of drug-resistant parasites and/or insecticide-resistant mosquito vectors necessitates developing alternative tools that either supplement or replace the conventional malaria control strategies. Trans-infecting the mosquito vector with symbionts that can either compete with a targeted pathogen or manipulate the host biology by reducing its vectorial capacity could be a promising and innovative biological approach for the control of infectious diseases This idea could be utilized to develop a novel and efficient vector control strategy; symbionts are dispersed into vector populations to reduce their ability to transmit human pathogens. Here, we reported the natural existence of Microsporidian (an obligate fungus) in the field-collected An. stephensi mosquito. However, laboratory-reared An. stephensi and An. culicifacies did not exhibit microsporidian infection. Similarly, 16s rRNA PCR identified ∼1kb amplicons in laboratory-reared An. stephensi and An. culicifacies, indicating the presence of naturally residing different bacterial species. DNA sequencing of these amplicons revealed the identities of different bacteria which are not well-characterized in terms of plasmodia-interaction activity in the Indian malaria vector. This article summarizes an overview of the previously studied microbial symbionts for their role in Plasmodium transmission along with a list of new or unexplored symbionts in the disease transmitting mosquito vectors. The summarized information could be utilized to explore such microbial symbionts for their role in Plasmodium-transmission biology in-depth and implementation in the malaria control interventions globally.