Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito.

Long-acting injectable medications, such as atovaquone, offer the prospect of a "chemical vaccine" for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field.

The Plasmodium falciparum apicoplast cysteine desulfurase provides sulfur for both iron-sulfur cluster assembly and tRNA modification.

Iron-sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite Plasmodium falciparum. Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.

Creating and Validating Ligase Primers to Detect Single Nucleotide Polymorphisms Associated with Atovaquone Resistance in Plasmodium falciparum.

Atovaquone-proguanil is one of the most commonly prescribed malaria prophylactic drugs. However, sporadic mutations conferring resistance to atovaquone have been detected in recent years associated with single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum cytochrome b ( pfcytb) gene. Monitoring polymorphisms linked with resistance is essential in assessing the prevalence of drug resistance and may help in designing strategies for malaria control. Several approaches have been used to study genetic polymorphisms associated with antimalarial drug resistance. However, they either lack high throughput capacity or are expensive in time or money. Ligase detection reaction fluorescent microsphere assay (LDR-FMA) provides a high-throughput method to detect genetic polymorphisms in P. falciparum. In this study, we have created primers to detect SNPs associated with clinically relevant atovaquone resistance using LDR-FMA and validated them in clinical samples. Four SNPs from pfcytb gene were analyzed using LDR-FMA. The results were 100% consistent with DNA sequence data, indicating that this method has potential as a tool to detect genetic polymorphisms associated with atovaquone resistance in P. falciparum.

Metabolic responses in blood-stage malaria parasites associated with increased and decreased sensitivity to PfATP4 inhibitors.

BACKGROUND: Spiroindolone and pyrazoleamide antimalarial compounds target Plasmodium falciparum P-type ATPase (PfATP4) and induce disruption of intracellular Na+ homeostasis. Recently, a PfATP4 mutation was discovered that confers resistance to a pyrazoleamide while increasing sensitivity to a spiroindolone. Transcriptomic and metabolic adaptations that underlie this seemingly contradictory response of P. falciparum to sublethal concentrations of each compound were examined to understand the different cellular accommodation to PfATP4 disruptions. METHODS: A genetically engineered P. falciparum Dd2 strain (Dd2A211V) carrying an Ala211Val (A211V) mutation in PfATP4 was used to identify metabolic adaptations associated with the mutation that results in decreased sensitivity to PA21A092 (a pyrazoleamide) and increased sensitivity to KAE609 (a spiroindolone). First, sublethal doses of PA21A092 and KAE609 causing substantial reduction (30-70%) in Dd2A211V parasite replication were identified. Then, at this sublethal dose of PA21A092 (or KAE609), metabolomic and transcriptomic data were collected during the first intraerythrocytic developmental cycle. Finally, the time-resolved data were integrated with a whole-genome metabolic network model of P. falciparum to characterize antimalarial-induced physiological adaptations. RESULTS: Sublethal treatment with PA21A092 caused significant (p < 0.001) alterations in the abundances of 91 Plasmodium gene transcripts, whereas only 21 transcripts were significantly altered due to sublethal treatment with KAE609. In the metabolomic data, a substantial alteration (≥ fourfold) in the abundances of carbohydrate metabolites in the presence of either compound was found. The estimated rates of macromolecule syntheses between the two antimalarial-treated conditions were also comparable, except for the rate of lipid synthesis. A closer examination of parasite metabolism in the presence of either compound indicated statistically significant differences in enzymatic activities associated with synthesis of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. CONCLUSION: The results of this study suggest that malaria parasites activate protein kinases via phospholipid-dependent signalling in response to the ionic perturbation induced by the Na+ homeostasis disruptor PA21A092. Therefore, targeted disruption of phospholipid signalling in PA21A092-resistant parasites could be a means to block the emergence of resistance to PA21A092.

Transmissibility of clinically relevant atovaquone-resistant Plasmodium falciparum by anopheline mosquitoes.

Rising numbers of malaria cases and deaths underscore the need for new interventions. Long-acting injectable medications, such as those now in use for HIV prophylaxis, offer the prospect of a malaria "chemical vaccine", combining the efficacy of a drug (like atovaquone) with the durability of a biological vaccine. Of concern, however, is the possible selection and transmission of drug-resistant parasites. We addressed this question by generating clinically relevant, highly atovaquone-resistant, Plasmodium falciparum mutants competent to infect mosquitoes. Isogenic paired strains, that differ only by a single Y268S mutation in cytochrome b, were evaluated in parallel in southeast Asian (Anopheles stephensi) or African (Anopheles gambiae) mosquitoes, and thence in humanized mice. Fitness costs of the mutation were evident along the lifecycle, in asexual parasite growth in vitro and in a progressive loss of parasites in the mosquito. In numerous independent experiments, microscopic exam of salivary glands from hundreds of mosquitoes failed to detect even one Y268S sporozoite, a defect not rescued by coinfection with wild type parasites. Furthermore, despite uniformly successful transmission of wild type parasites from An. stephensi to FRG NOD huHep mice bearing human hepatocytes and erythrocytes, multiple attempts with Y268S-fed mosquitoes failed: there was no evidence of parasites in mouse tissues by microscopy, in vitro culture, or PCR. These studies confirm a severe-to-lethal fitness cost of clinically relevant atovaquone-resistant P. falciparum in the mosquito, and they significantly lessen the likelihood of their transmission in the field.

New insights into apicoplast metabolism in blood-stage malaria parasites.

The apicoplast of Plasmodium falciparum is the only source of essential isoprenoid precursors and Coenzyme A (CoA) in the parasite. Isoprenoid precursor synthesis relies on the iron-sulfur cluster (FeS) cofactors produced within the apicoplast, rendering FeS synthesis an essential function of this organelle. Recent reports provide important insights into the roles of FeS cofactors and the use of isoprenoid precursors and CoA both inside and outside the apicoplast. Here, we review the recent insights into the roles of these metabolites in blood-stage malaria parasites and discuss new questions that have been raised in light of these discoveries.

Screening the Pathogen Box for Inhibition of Plasmodium falciparum Sporozoite Motility Reveals a Critical Role for Kinases in Transmission Stages.

As the malaria parasite becomes resistant to every drug that we develop, the identification and development of novel drug candidates are essential. Many studies have screened compounds designed to target the clinically important blood stages. However, if we are to shrink the malaria map, new drugs that block the transmission of the parasite are needed. Sporozoites are the infective stage of the malaria parasite, transmitted to the mammalian host as mosquitoes probe for blood. Sporozoite motility is critical to their ability to exit the inoculation site and establish infection, and drug-like compounds targeting motility are effective at blocking infection in the rodent malaria model. In this study, we established a moderate-throughput motility assay for sporozoites of the human malaria parasite Plasmodium falciparum, enabling us to screen the 400 drug-like compounds from the pathogen box provided by the Medicines for Malaria Venture for their activity. Compounds exhibiting inhibitory effects on P. falciparum sporozoite motility were further assessed for transmission-blocking activity and asexual-stage growth. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility in the nanomolar range. Using membrane feeding assays, we demonstrate that four of these compounds had inhibitory activity against the transmission of P. falciparum to the mosquito. Interestingly, of the four compounds with inhibitory activity against both transmission stages, three are known kinase inhibitors. Together with a previous study that found that several of these compounds could inhibit asexual blood-stage parasite growth, our findings provide new antimalarial drug candidates that have multistage activity.

The ferredoxin redox system - an essential electron distributing hub in the apicoplast of Apicomplexa.

The apicoplast, a relict plastid found in most species of the phylum Apicomplexa, harbors the ferredoxin redox system which supplies electrons to enzymes of various metabolic pathways in this organelle. Recent reports in Toxoplasma gondii and Plasmodium falciparum have shown that the iron-sulfur cluster (FeS)-containing ferredoxin is essential in tachyzoite and blood-stage parasites, respectively. Here we review ferredoxin's crucial contribution to isoprenoid and lipoate biosynthesis as well as tRNA modification in the apicoplast, highlighting similarities and differences between the two species. We also discuss ferredoxin's potential role in the initial reductive steps required for FeS synthesis as well as recent evidence that offers an explanation for how NADPH required by the redox system might be generated in Plasmodium spp.

Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure.

Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused a significant reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol-a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

Roles of Ferredoxin-Dependent Proteins in the Apicoplast of Plasmodium falciparum Parasites.

Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form a redox system that is hypothesized to play a central role in the maintenance and function of the apicoplast organelle of malaria parasites. The Fd/FNR system provides reducing power to various iron-sulfur cluster (FeS)-dependent proteins in the apicoplast and is believed to help to maintain redox balance in the organelle. While the Fd/FNR system has been pursued as a target for antimalarial drug discovery, Fd, FNR, and the FeS proteins presumably reliant on their reducing power play an unknown role in parasite survival and apicoplast maintenance. To address these questions, we generated genetic deletions of these proteins in a parasite line containing an apicoplast bypass system. Through these deletions, we discovered that Fd, FNR, and certain FeS proteins are essential for parasite survival but found that none are required for apicoplast maintenance. Additionally, we addressed the question of how Fd and its downstream FeS proteins obtain FeS cofactors by deleting the FeS transfer proteins SufA and NfuApi. While individual deletions of these proteins revealed their dispensability, double deletion resulted in synthetic lethality, demonstrating a redundant role in providing FeS clusters to Fd and other essential FeS proteins. Our data support a model in which the reducing power from the Fd/FNR system to certain downstream FeS proteins is essential for the survival of blood-stage malaria parasites but not for organelle maintenance, while other FeS proteins are dispensable for this stage of parasite development. IMPORTANCE Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form one of the few known redox systems in the apicoplast of malaria parasites and provide reducing power to iron-sulfur (FeS) cluster proteins within the organelle. While the Fd/FNR system has been explored as a drug target, the essentiality and roles of this system and the identity of its downstream FeS proteins have not been determined. To answer these questions, we generated deletions of these proteins in an apicoplast metabolic bypass line (PfMev) and determined the minimal set of proteins required for parasite survival. Moving upstream of this pathway, we also generated individual and dual deletions of the two FeS transfer proteins that deliver FeS clusters to Fd and downstream FeS proteins. We found that both transfer proteins are dispensable, but double deletion displayed a synthetic lethal phenotype, demonstrating their functional redundancy. These findings provide important insights into apicoplast biochemistry and drug development.

Persistence of Markers of Chloroquine Resistance in Plasmodium falciparum in Bangladesh.

The control of malaria, in terms of drug resistance, remains a significant global challenge, with Bangladesh, a malaria-endemic country, being no exception. The aim of this study was to explore antimalarial resistance in Bangladesh by molecular analysis of Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance transporter 1 (pfmdr1) genetic markers of P. falciparum. Samples were obtained from uncomplicated malaria patients between 2009 and 2014 from six malaria-endemic districts. Based on parasite transmission intensity, the endemic districts were divided into high-transmission (Chittagong Hill Tracts [CHT]) and low-transmission (non-CHT) regions. Falciparum malaria-positive isolates were genotyped for K76T of the pfcrt gene, and N86Y and Y184F of the pfmdr1 gene: in total, 262 P. falciparum clinical isolates were analyzed. In CHT areas, the prevalence of polymorphisms was 70.6% for 76T, 14.4% for 86Y, and 7.8% for 184F. In non-CHT areas, 76T and 86Y mutations were found in 78.0% and 19.5% of the samples, respectively, whereas no 184F mutations were observed. We compared our data with previous similar molecular observations, which shows a significant decrease in pfcrt 76T mutation prevalence. No pfmdr1 amplification was observed in any of the samples suggesting an unaltered susceptibility to amino alcohol drugs such as mefloquine and lumefantrine. This study provides an updated assessment of the current status of pfcrt and pfmdr1 gene mutations in Bangladesh, and suggests there is persistent high prevalence of markers of resistance to aminoquinoline drugs.

Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparum.

Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/ß hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.

Internalization of Erythrocyte Acylpeptide Hydrolase Is Required for Asexual Replication of Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum causes disease as it replicates within the host's erythrocytes. We have found that an erythrocyte serine hydrolase, acylpeptide hydrolase (APEH), accumulates within developing asexual parasites. Internalization of APEH was associated with a proteolytic event that reduced the size of the catalytic polypeptide from 80 to 55 kDa. A triazole urea APEH inhibitor, termed AA74-1, was employed to characterize the role of parasite-internalized APEH. In cell lysates, AA74-1 was a potent and highly selective inhibitor of both host erythrocyte and parasite-internalized APEH. When added to cultures of ring-stage parasites, AA74-1 was a poor inhibitor of replication over one asexual replication cycle; however, its potency increased dramatically after a second cycle. This enhancement of potency was not abrogated by the addition of exogenous isopentenyl pyrophosphate, the sole essential product of apicoplast metabolism. High-potency inhibition of parasite growth could be effected by adding AA74-1 to schizont-stage parasites, which resulted in parasite death at the early trophozoite stage of the ensuing replication cycle. Analysis of APEH inhibition in intact cultured cells revealed that host erythrocyte APEH, but not the parasite-internalized APEH pool, was inhibited by exogenous AA74-1. Our data support a model for the mode of parasiticidal activity of AA74-1 whereby sustained inactivation of host erythrocyte APEH is required prior to merozoite invasion and during parasite asexual development. Together, these findings provide evidence for an essential catalytic role for parasite-internalized APEH.IMPORTANCE Nearly half a million deaths were attributed to malaria in 2017. Protozoan parasites of the genus Plasmodium cause disease in humans while replicating asexually within the host's erythrocytes, with P. falciparum responsible for most of the mortality. Understanding how Plasmodium spp. have adapted to their unique host erythrocyte environment is important for developing malaria control strategies. Here, we demonstrate that P. falciparum coopts a host erythrocyte serine hydrolase termed acylpeptide hydrolase. By showing that the parasite requires acylpeptide hydrolase activity for replication, we expand our knowledge of host cell factors that contribute to robust parasite growth.

Biological Studies and Target Engagement of the 2- C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase (IspD)-Targeting Antimalarial Agent (1 R,3 S)-MMV008138 and Analogs.

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2- C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure-activity relationships by probing the ability of MMV008138 analogs to inhibit PfIspD recombinant enzyme. Here, we report PfIspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and PfIspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents.

Abundance and Dynamics of Anopheles (Diptera: Culicidae) Larvae in a Malaria Endemic Area of Bangladesh.

Malaria is a major health burden in the border-belt areas of Bangladesh. There are recent data from adult mosquito collections that a number of vectors are involved in the transmission cycle. However, little information regarding the larval habitats of Anopheles mosquitoes are available in Bangladesh. To start filling this gap, a cross-sectional larval survey was conducted in Bandarban, Bangladesh from October 2011 to September 2012. Descriptive analysis, Poisson regression, spearman correlations and zero-inflated Poisson regression were used to calculate the degree of association between the abundance of larval Anopheles species and environmental factors. From the 300 larval habitats sampled, 5,568 Anopheles larvae were collected and of these, 2,263 (40.6%) were identified to species. Collections represented 16 Anopheles species with Anopheles vagus (26.4%, n = 598) as the dominant species. A total of 16 Anopheles larval habitat types were identified. Larval abundance was significantly different (P < 0.05) among habitats with pond (40%, n = 914) and rice field (34%, n = 779) implicated to be the most utilized. Larval abundance varied significantly (P < 0.05) with habitat characteristics. Most of the larvae were collected from sites with a range of pH from 7.0 to 8.0. Data obtained from this study revealed both natural and human-created larval habitats were favorable for anopheline larval survival and development. Such information elucidates plausible drivers of high anopheline diversity, high vector abundance, changes in relative species abundance from historic data, and sustained transmission of malaria in endemic areas of Bangladesh.

Plasmodium falciparum Genetic Diversity in Bangladesh Does Not Suggest a Hypoendemic Population Structure.

Despite the recommendation for the use of merozoite surface protein 1 (msp1), merozoite surface protein 2 (msp2), and glutamate-rich protein (glurp) genes as markers in drug efficacy studies by World Health Organization and their limited use in Bangladesh, the circulating Plasmodium falciparum population genetic structure has not yet been assessed in Bangladesh. This study presents a comprehensive report on the circulating P. falciparum population structure based on msp1, msp2, and glurp polymorphic gene markers in Bangladesh. Among the 130 pretreatment (day 0) P. falciparum samples from seven malaria-endemic districts, 14 distinct genotypes were observed for msp1, 20 for msp2, and 13 for glurp Polyclonal infection was reported in 94.6% (N = 123) of the samples. Multiplicity of infection (MOI) for msp1 was the highest (1.5) among the MOIs of the markers. The heterozygosity for msp1, msp2, and glurp was 0.89, 0.93, and 0.83, respectively. Data according to different malaria-endemic areas are also presented and discussed. Bangladesh is considered as a malaria-hypoendemic country. However, the prevalence of polyclonal infection and the genetic diversity of P. falciparum do not represent hypoendemicity.

Genetic diversity of Plasmodium vivax in clinical isolates from Bangladesh.

BACKGROUND: Plasmodium vivax is the second most prevalent human malaria parasite in Bangladesh; however, there are no data of its genetic diversity. Several molecular markers are available where Pvcsp, Pvmsp 1 and Pvmsp 3α are most commonly used for P. vivax genotyping studies. The aim of the study was to investigate the population structure of P. vivax in Bangladesh. METHODS: A total of 102 P. vivax-positive blood samples were collected from different malaria-endemic areas in Bangladesh and subsequently analysed for those three genotyping markers. Nested PCR was performed for diagnosis and genotyping analysis followed by PCR-RFLP to detect genetic diversity using Pvcsp, Pvmsp 1 and Pvmsp 3α markers. RESULTS: Analysis of Pvcsp showed that the VK210 repeat type was highly prevalent (64.7%, 66/102) compared to VK247 (35.3%, 36/102), although the prevalence of VK247 was higher than other Southeast Asian countries. Analysis of these three genes revealed a diverse, circulating population of P. vivax where a total of ten, 56 and 35 distinct genotypes were detected for Pvcsp, Pvmsp 1 and Pvmsp 3α, respectively. CONCLUSION: This genotyping observation of P. vivax is the first report from Bangladesh and will provide valuable information for establishing the genotyping methods and circulating genetic variants of these three markers available in Bangladesh.

SEX DETERMINATION. A male-determining factor in the mosquito Aedes aegypti.

Sex determination in the mosquito Aedes aegypti is governed by a dominant male-determining factor (M factor) located within a Y chromosome-like region called the M locus. Here, we show that an M-locus gene, Nix, functions as an M factor in A. aegypti. Nix exhibits persistent M linkage and early embryonic expression, two characteristics required of an M factor. Nix knockout with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 resulted in largely feminized genetic males and the production of female isoforms of two key regulators of sexual differentiation: doublesex and fruitless. Ectopic expression of Nix resulted in genetic females with nearly complete male genitalia. Thus, Nix is both required and sufficient to initiate male development. This study provides a foundation for mosquito control strategies that convert female mosquitoes into harmless males.

Role of underappreciated vectors in malaria transmission in an endemic region of Bangladesh-India border.

BACKGROUND: Despite the efforts of the National Malaria Control Programme, malaria remains as an important public health problem in Bangladesh, particularly in the south-eastern region bordering India. Successful malaria control strategies rely on a detailed understanding of the underlying causes of malaria transmission. Here, an entomological survey was conducted in a malaria endemic area of Bangladesh bordering India to investigate the Anopheles mosquito community and assess their Plasmodium infection status. METHODS: Monthly entomological collections were undertaken from October 2010 to September 2011 in five villages in the Matiranga sub-district, Khagrachari district in Bangladesh, bordering the Indian State of Tripura. CDC miniature light traps were placed inside houses to collect adult Anopheles mosquitoes. Following morphological and molecular identification of the female Anopheles mosquitoes collected, they were screened for circumsporozoite proteins (CSP) of Plasmodium falciparum (Pf), Plasmodium vivax-210 (Pv-210) and Plasmodium vivax-247 (Pv-247), by ELISA to determine natural infection rates. Variation in Anopheles species composition, relative abundance and Plasmodium infection rates were analysed between sampled villages. RESULTS: A total of 2,027 female Anopheles were collected, belonging to 20 species. Anopheles nivipes was the most abundant species in our test villages during the peak malaria transmission season, and was observed sympatrically with An. philippinensis in the studied area. However, in the dry off-peak season, An. jeyporiensis was the most abundant species. Shannon's diversity index was highest in October (2.12) and evenness was highest in May (0.91). The CSP ELISA positive rate overall was 0.44%. Anopheles karwari (n=2), An. barbirostris s.l. (n=1) and An. vagus (n=1) were recorded positive for Pf. Anopheles kochi (n=1) was positive for Pv-210 while An. umbrosus (n=1), An. nivipes (n=1) and An. kochi (n=1) were positive for Pv-247. A mixed infection of Pf and Pv-247 was detected in An. barbirostris s.l.. CONCLUSION: High diversity of Anopheles species was observed in areas close to the international border where species that were underestimated for malaria transmission significantly outnumbered principal vector species and these may play a significantly heightened role in malaria transmission.

A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria.

Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5parasites/µL of infected blood within 35min, while the other LAMP method tested in this study, could detect a minimum of 100parasites/µL of human blood after 60min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories.