Genetic analysis of the orthologous crt and mdr1 genes in Plasmodium malariae from Thailand and Myanmar.

BACKGROUND: Plasmodium malariae is a widely spread but neglected human malaria parasite, which causes chronic infections. Studies on genetic polymorphisms of anti-malarial drug target genes in P. malariae are limited. Previous reports have shown polymorphisms in the P. malariae dihydrofolate reductase gene associated with pyrimethamine resistance and linked to pyrimethamine drug pressure. This study investigated polymorphisms of the P. malariae homologous genes, chloroquine resistant transporter and multidrug resistant 1, associated with chloroquine and mefloquine resistance in Plasmodium falciparum. METHODS: The orthologous P. malariae crt and mdr1 genes were studied in 95 patients with P. malariae infection between 2002 and 2016 from Thailand (N = 51) and Myanmar (N = 44). Gene sequences were analysed using BioEdit, MEGA7, and DnaSP programs. Mutations and gene amplifications were compared with P. falciparum and Plasmodium vivax orthologous genes. Protein topology models derived from the observed pmcrt and pmmdr1 haplotypes were constructed and analysed using Phyre2, SWISS MODEL and Discovery Studio Visualization V 17.2. RESULTS: Two non-synonymous mutations were observed in exon 2 (H53P, 40%) and exon 8 (E278D, 44%) of pmcrt. The topology model indicated that H53P and E278D were located outside of the transmembrane domain and were unlikely to affect protein function. Pmmdr1 was more diverse than pmcrt, with 10 non-synonymous and 3 synonymous mutations observed. Non-synonymous mutations were located in the parasite cytoplasmic site, transmembrane 11 and nucleotide binding domains 1 and 2. Polymorphisms conferring amino acid changes in the transmembrane and nucleotide binding domains were predicted to have some effect on PmMDR1 conformation, but were unlikely to affect protein function. All P. malariae parasites in this study contained a single copy of the mdr1 gene. CONCLUSIONS: The observed polymorphisms in pmcrt and pmmdr1 genes are unlikely to affect protein function and unlikely related to chloroquine drug pressure. Similarly, the absence of pmmdr1 copy number variation suggests limited mefloquine drug pressure on the P. malariae parasite population, despite its long time use in Thailand for the treatment of falciparum malaria.

Assessment of effectiveness of DAMaN: A malaria intervention program initiated by Government of Odisha, India.

India, a persistently significant contributor to the global malaria burden, rolled out several anti-malaria interventions at the national and state level to control and recently, to eliminate the disease. Odisha, the eastern Indian state with the highest malaria burden experienced substantial gains shown by various anti-malaria initiatives implemented under the National Vector-borne Disease Control Programme (NVBDCP). However, recalcitrant high-transmission "pockets" of malaria persist in hard-to-reach stretches of the state, characterised by limited access to routine malaria surveillance and the forested hilly topography favouring unbridled vector breeding. The prevalence of asymptomatic malaria in such pockets serves as perpetual malaria reservoir, thus hindering its elimination. Therefore, a project with the acronym DAMaN was initiated since 2017 by state NVBDCP, targeting locally identified high endemic 'pockets' in 23 districts. DAMaN comprised biennial mass screening and treatment, provisioning of long-lasting insecticidal net (LLIN) and behavioural change communication. Subsequently, to inform policy, assessment of DAMaN was conceived that aims to estimate the coverage of the various components of the project; the prevalence of malaria, even at sub-patent level especially among pregnant/lactating women and children; and its impact on malaria incidence. A survey of DAMaN beneficiaries will measure coverage; and knowledge and practices related to LLIN; along with collection of blood specimens from a probability sample. A multi-stage stratified clustered sample of 2228 households (~33% having pregnant/lactating women) will be selected from 6 DAMaN districts. Routine DAMaN project data (2017-2018) and NVBDCP data (2013-2018) will be extracted. Rapid Diagnostic Test, Polymerase Chain Reaction and blood smear microscopy will be conducted to detect malarial parasitemia. In addition to measuring DAMaN's coverage and malarial prevalence in DAMaN pockets, its impact will be estimated using pre-post differences and Interrupted Time Series analysis using 2017 as the "inflection" point. The assessment may help to validate the unique strategies employed by DAMaN.

Open questions for harnessing autophagy-modulating drugs in the SARS-CoV-2 war: hope or hype?

At a time when the world faces an emotional breakdown, crushing our dreams, if not, taking our lives, we realize that together we must fight the war against the COVID-19 outbreak even if almost the majority of the scientific community finds itself confined at home. Every day, we, scientists, listen to the latest news with its promises and announcements. Across the world, a surge of clinical trials trying to cure or slow down the coronavirus pandemic has been launched to bring hope instead of fear and despair. One first proposed clinical trial has drawn worldwide hype to the benefit of chloroquine (CQ), in the treatment of patients infected by the recently emerged deadly coronavirus (SARS-CoV-2). We should consider this information in light of the long-standing anti-inflammatory and anti-viral properties of CQ-related drugs. Yet, none of the articles promoting the use of CQ in the current pandemic evoked a possible molecular or cellular mechanism of action that could account for any efficacy. Here, given the interaction of viruses with macroautophagy (hereafter referred to as autophagy), a CQ-sensitive anti-viral safeguard pathway, we would like to discuss the pros, but also the cons concerning the current therapeutic options targeting this process.

Screening Marine Natural Products for New Drug Leads against Trypanosomatids and Malaria.

Neglected Tropical Diseases (NTD) represent a serious threat to humans, especially for those living in poor or developing countries. Almost one-sixth of the world population is at risk of suffering from these diseases and many thousands die because of NTDs, to which we should add the sanitary, labor and social issues that hinder the economic development of these countries. Protozoan-borne diseases are responsible for more than one million deaths every year. Visceral leishmaniasis, Chagas disease or sleeping sickness are among the most lethal NTDs. Despite not being considered an NTD by the World Health Organization (WHO), malaria must be added to this sinister group. Malaria, caused by the apicomplexan parasite Plasmodium falciparum, is responsible for thousands of deaths each year. The treatment of this disease has been losing effectiveness year after year. Many of the medicines currently in use are obsolete due to their gradual loss of efficacy, their intrinsic toxicity and the emergence of drug resistance or a lack of adherence to treatment. Therefore, there is an urgent and global need for new drugs. Despite this, the scant interest shown by most of the stakeholders involved in the pharmaceutical industry makes our present therapeutic arsenal scarce, and until recently, the search for new drugs has not been seriously addressed. The sources of new drugs for these and other pathologies include natural products, synthetic molecules or repurposing drugs. The most frequent sources of natural products are microorganisms, e.g., bacteria, fungi, yeasts, algae and plants, which are able to synthesize many drugs that are currently in use (e.g. antimicrobials, antitumor, immunosuppressants, etc.). The marine environment is another well-established source of bioactive natural products, with recent applications against parasites, bacteria and other pathogens which affect humans and animals. Drug discovery techniques have rapidly advanced since the beginning of the millennium. The combination of novel techniques that include the genetic modification of pathogens, bioimaging and robotics has given rise to the standardization of High-Performance Screening platforms in the discovery of drugs. These advancements have accelerated the discovery of new chemical entities with antiparasitic effects. This review presents critical updates regarding the use of High-Throughput Screening (HTS) in the discovery of drugs for NTDs transmitted by protozoa, including malaria, and its application in the discovery of new drugs of marine origin.

The antimalarial screening landscape-looking beyond the asexual blood stage.

In recent years, the research agenda to tackle global morbidity and mortality from malaria disease has shifted towards innovation, in the hope that efforts at the frontiers of scientific research may re-invigorate gains made towards eradication. Discovery of new antimalarial drugs with novel chemotypes or modes of action lie at the heart of these efforts. There is a particular interest in drug candidates that target stages of the malaria parasite lifecycle beyond the symptomatic asexual blood stages. This is especially important given the spectre of emerging drug resistance to all current frontline antimalarials. One approach gaining increased interest is the potential of designing novel drugs that target parasite passage from infected individual to feeding mosquito and back again. Action of such therapeutics is geared much more at the population level rather than just concerned with the infected individual. The search for novel drugs active against these stages has been helped by improvements to in vitro culture of transmission and pre-erythrocytic parasite lifecycle stages, robotic automation and high content imaging, methodologies that permit the high-throughput screening (HTS) of compound libraries for drug discovery. Here, we review recent advances in the antimalarial screening landscape, focussed on transmission blocking as a key aim for drug-treatment campaigns of the future.

Malaria Elimination: Time to Target All Species.

Important strides have been made within the past decade toward malaria elimination in many regions, and with this progress, the feasibility of eradication is once again under discussion. If the ambitious goal of eradication is to be achieved by 2040, all species of Plasmodium infecting humans will need to be targeted with evidence-based and concerted interventions. In this perspective, the potential barriers to achieving global malaria elimination are discussed with respect to the related diversities in host, parasite, and vector populations. We argue that control strategies need to be reorientated from a sequential attack on each species, dominated by Plasmodium falciparum to one that targets all species in parallel. A set of research themes is proposed to mitigate the potential setbacks on the pathway to a malaria-free world.

Prospective Clinical Trial Assessing Species-Specific Efficacy of Artemether-Lumefantrine for the Treatment of Plasmodium malariae, Plasmodium ovale, and Mixed Plasmodium Malaria in Gabon.

Treatment recommendations for Plasmodium malariae and Plasmodium ovale malaria are largely based on anecdotal evidence. The aim of this prospective study, conducted in Gabon, was to systematically assess the efficacy and safety of artemether-lumefantrine for the treatment of patients with uncomplicated P. malariae or P. ovale species monoinfections or mixed Plasmodium infections. Patients with microscopically confirmed P. malariae, P. ovale, or mixed-species malaria with at least one of these two Plasmodium species were treated with an oral, fixed-dose combination of artemether-lumefantrine for 3 consecutive days. The primary endpoints were per-protocol PCR-corrected adequate clinical and parasitological response (ACPR) on days 28 and 42. Tolerability and safety were recorded throughout the follow-up period. Seventy-two participants (42 male and 30 female) were enrolled; 62.5% of them had PCR-corrected mixed Plasmodium infections. Per protocol, PCR-corrected ACPR rates were 96.6% (95% confidence interval [CI], 91.9 to 100) on day 28 and 94.2% (95% CI, 87.7 to 100) on day 42. Considering Plasmodium species independently from their coinfecting species, day 42 ACPR rates were 95.5% (95% CI, 89.0 to 100) for P. falciparum, 100% (exact CI, 84.6 to 100) for P. malariae, 100% (exact CI, 76.8 to 100) for P. ovale curtisi, and 90.9% (95% CI, 70.7 to 100) for P. ovale wallikeri Study drug-related adverse events were generally mild or moderate. In conclusion, this clinical trial demonstrated satisfying antimalarial activity of artemether-lumefantrine against P. ovalewallikeri, P. ovale curtisi, P. malariae, and mixed Plasmodium infections, with per-protocol efficacies of 90% to 100% and without evident tolerability or safety concerns. (This trial was registered in the clinical study database ClinicalTrials.gov under the identifier NCT02528279.).

Clinical implications of a gradual dormancy concept in malaria.

Malaria recurrences after an initially successful therapy and malarial fever occurring a long time after infection are well-known problems in malariology. Currently, two distinct types of malaria recurrences are defined: recrudescence and relapse. A recrudescence is thought to originate from circulating Plasmodium blood stages which do not cause fever before a certain level of a microscopically detectable parasitemia is reached. Contrary, a relapse is thought to originate from quiescent intracellular hepatic parasite stages called hypnozoites. Recrudescences would typically occur in infections due to Plasmodium falciparum. Plasmodium knowlesi, and Plasmodium malariae, whereas relapses would be caused exclusively by Plasmodium vivax and Plasmodium ovale. This schematic view is, however, insufficiently supported by experimental evidence. For instance, hypnozoites of P. ovale have never been experimentally documented. On the other hand, the nonfinding of P. malariae hypnozoites turned into the proof for the nonexistence of P. malariae hypnozoites. Clinical relapse-type recurrences have been observed in both P. ovale and P. malariae infections, and decade-long incubation times have also been reported in P. falciparum infections. We propose a gradual hypothesis in accordance with the continuity concept of biological evolution: both, relapse and recrudescence may be potentially caused by all Plasmodium spp. We hypothesize that the difference between the various Plasmodium spp. is quantitative rather than qualitative: there are Plasmodium spp. which frequently cause relapses such as P. vivax, particularly the P.v. Chesson strain, species which cause relapses less frequently, such as P. ovale and sometimes P. malariae, and species which may exceptionally cause relapses such as P. falciparum. All species may cause recrudescences. As clinical consequences, we propose that 8-aminquinolines may be considered in a relapse-type recrudescence regardless of the causal Plasmodium sp., whereas primaquine relapse prevention might not be routinely indicated in malaria due to P. ovale.

Limited Polymorphism of the Kelch Propeller Domain in Plasmodium malariae and P. ovale Isolates from Thailand.

Artemisinin resistance in Plasmodium falciparum, the agent of severe malaria, is currently a major obstacle to malaria control in Southeast Asia. A gene named "kelch13" has been associated with artemisinin resistance in P. falciparum The orthologue of the kelch gene in P. vivax was identified and a small number of mutations were found in previous studies. The kelch orthologues in the other two human malaria parasites, P. malariae and P. ovale, have not yet been studied. Therefore, in this study, the orthologous kelch genes of P. malariae, P. ovale wallikeri, and P. ovale curtisi were isolated and analyzed for the first time. The homologies of the kelch genes of P. malariae and P. ovale were 84.8% and 82.7%, respectively, compared to the gene in P. falciparum kelch polymorphisms were studied in 13 P. malariae and 5 P. ovale isolates from Thailand. There were 2 nonsynonymous mutations found in these samples. One mutation was P533L, which was found in 1 of 13 P. malariae isolates, and the other was K137R, found in 1 isolate of P. ovale wallikeri (n = 4). This result needs to be considered in the context of widespread artemisinin used within the region; their functional consequences for artemisinin sensitivity in P. malariae and P. ovale will need to be elucidated.

Delayed Onset of Symptoms and Atovaquone-Proguanil Chemoprophylaxis Breakthrough by Plasmodium malariae in the Absence of Mutation at Codon 268 of pmcytb.

Plasmodium malariae is widely distributed across the tropics, causing symptomatic malaria in humans with a 72-hour fever periodicity, and may present after latency periods lasting up to many decades. Delayed occurrence of symptoms is observed in humans using chemoprophylaxis, or patients having received therapies targeting P. falciparum intraerythrocytic asexual stages, but few investigators have addressed the biological basis of the ability of P. malariae to persist in the human host. To investigate these interesting features of P. malariae epidemiology, we assembled, here, an extensive case series of P. malariae malaria patients presenting in non-endemic China, Sweden, and the UK who returned from travel in endemic countries, mainly in Africa. Out of 378 evaluable P. malariae cases, 100 (26.2%) reported using at least partial chemoprophylaxis, resembling the pattern seen with the relapsing parasites P. ovale spp. and P. vivax. In contrast, for only 7.5% of imported UK cases of non-relapsing P. falciparum was any chemoprophylaxis use reported. Genotyping of parasites from six patients reporting use of atovaquone-proguanil chemoprophylaxis did not reveal mutations at codon 268 of the cytb locus of the P. malariae mitochondrial genome. While travellers with P. malariae malaria are significantly more likely to report prophylaxis use during endemic country travel than are those with P. falciparum infections, atovaquone-proguanil prophylaxis breakthrough was not associated with pmcytb mutations. These preliminary studies, together with consistent observations of the remarkable longevity of P. malariae, lead us to propose re-examination of the dogma that this species is not a relapsing parasite. Further studies are needed to investigate our favoured hypothesis, namely that P. malariae can initiate a latent hypnozoite developmental programme in the human hepatocyte: if validated this will explain the consistent observations of remarkable longevity of parasitism, even in the presence of antimalarial prophylaxis or treatment.

Novel Cross-Border Approaches to Optimise Identification of Asymptomatic and Artemisinin-Resistant Plasmodium Infection in Mobile Populations Crossing Cambodian Borders.

BACKGROUND: Human population movement across country borders presents a real challenge for malaria control and elimination efforts in Cambodia and its neighbouring countries. To quantify Plasmodium infection among the border-crossing population, including asymptomatic and artemisinin resistant (AR) parasites, three official border crossing points, one from each of Cambodia's borders with Thailand, Laos and Vietnam, were selected for sampling. METHODS AND FINDINGS: A total of 3206 participants (of 4110 approached) were recruited as they crossed the border, tested for malaria and interviewed. By real-time polymerase chain reaction (RT-PCR), 5.4% of all screened individuals were found to harbour Plasmodium parasites. The proportion was highest at the Laos border (11.5%). Overall there were 97 P. vivax (55.7%), 55 P. falciparum (31.6%), two P. malariae (1.1%) and 20 mixed infections (11.5%). Of identified infections, only 20% were febrile at the time of screening. Of the 24 P. falciparum samples where a further PCR was possible to assess AR, 15 (62.5%) had mutations in the K13 propeller domain gene, all from participants at the Laos border point. Malaria rapid diagnostic test (RDT) pLDH/HRP-2 identified a positivity rate of 3.2% overall and sensitivity compared to RT-PCR was very low (43.1%). Main individual risk factors for infection included sex, fever, being a forest-goer, poor knowledge of malaria prevention methods and previous malaria infection. Occupation, day of the week and time of crossing (morning vs. afternoon) also appeared to play an important role in predicting positive cases. CONCLUSIONS: This study offers a novel approach to identify asymptomatic infections and monitor AR parasite flow among mobile and migrant populations crossing the borders. Similar screening activities are recommended to identify other hot borders and characterise potential hot spots of AR. Targeted "customised" interventions and surveillance activities should be implemented in these sites to accelerate elimination efforts in the region.

The rise and fall of malaria in a West African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study.

BACKGROUND: A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years. METHODS: Between 1990 and 2012, we did a prospective longitudinal study of the inhabitants of Dielmo, Senegal, to identify all episodes of fever and investigate the relation between malaria host, vector, and parasite. Our study included daily medical surveillance with systematic parasite detection in individuals with fever. We measured parasite prevalence four times a year with cross-sectional surveys. We monitored malaria transmission monthly with night collection of mosquitoes. Malaria treatment changed over the years, from quinine (1990-94), to chloroquine (1995-2003), amodiaquine plus sulfadoxine-pyrimethamine (2003-06), and finally artesunate plus amodiaquine (2006-12). Insecticide-treated nets (ITNs) were introduced in 2008. FINDINGS: We monitored 776 villagers aged 0-101 years for 2 378 150 person-days of follow-up. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012. Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. We recorded 23 546 fever episodes during the study, including 8243 clinical attacks caused by Plasmodium falciparum, 290 by Plasmodium malariae, and 219 by Plasmodium ovale. Three deaths were directly attributable to malaria, and two to severe adverse events of antimalarial drugs. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs. INTERPRETATION: Malaria control policies combining prompt treatment of clinical attacks and deployment of ITNs can nearly eliminate parasite carriage and greatly reduce the burden of malaria in populations exposed to intense perennial malaria transmission. The choice of drugs seems crucial. Rapid decline of clinical immunity allows rapid detection and treatment of novel infections and thus has a key role in sustaining effectiveness of combining artemisinin-based combination therapy and ITNs despite increasing pyrethroid resistance. FUNDING: Pasteur Institutes of Dakar and Paris, Institut de Recherche pour le Développement, and French Ministry of Cooperation.

Choline analogues in malaria chemotherapy.

Emerging resistance against well-established anti-malaria drugs warrants the introduction of new therapeutic agents with original mechanisms of action. Inhibition of membrane-based phospholipid biosynthesis, which is crucial for the parasite, has thus been proposed as a novel and promising therapeutic strategy. This review compiles literature concerning the design and study of choline analogues and related cation derivatives as potential anti-malarials. It covers advances achieved over the last two decades and describes: the concept validation, the design and selection of a clinical candidate (Albitiazolium), back-up derivatives while also providing insight into the development of prodrug approaches.

Towards histone deacetylase inhibitors as new antimalarial drugs.

Histone deacetylases (HDACs) are important enzymes that effect post-translational modifications of proteins by altering the acetylation state of lysine residues. HDACs control epigenetic changes that trigger cell transformation and proliferation of transformed cells associated with many diseases. These enzymes are validated drug targets for some types of cancer and are promising therapeutic targets for a range of other diseases, including malaria. Annually, there are ~500 million clinical cases of malaria and ~0.8-1.2 million deaths. There is no licensed vaccine for preventing malaria, and parasites that cause malaria are becoming resistant to current drugs, necessitating the search for new therapies. HDAC inhibitors are emerging as a promising new class of antimalarial drugs with potent and selective action against Plasmodium parasites in vitro. Recent studies on the effects of HDAC inhibitors on the growth and development of P. falciparum have provided important new information on transcriptional regulation in malaria parasites and have validated the potential of this class of inhibitors for malaria therapy. To realise effective HDAC inhibitors for clinical trials, next generation inhibitors must not inhibit other human HDACs or proteins required for normal human physiology, be highly selective in killing parasites in vivo without killing normal host cells, and have improved bioavailability and pharmacokinetic profiles. This review summarizes current knowledge about malaria parasite HDACs and HDAC inhibitors with antimalarial properties, and provides insights for their development into new drugs for treatment of malaria.

Novel drug targets in malaria parasite with potential to yield antimalarial drugs with long useful therapeutic lives.

The status of chemotherapy as the main strategy in malaria control is rapidly being eroded by development of drug resistant Plasmodia, causing malaria to be dubbed a "re-emerging disease". To counter this misfortune, there is an urgent need to develop novel antimalarial drugs capable of delaying resistance, or circumventing it altogether. Mode of action of antimalarial drugs, inter alia, has a bearing on their useful therapeutic lives (UTLs), with single target drugs having short UTLs compared with drugs which possess pleiotropic action. Quinolines and artemisinins are the two classes of drugs with pleiotropic action and subsequently long UTLs. All other antimalarials are single-target drugs, and have been rendered ineffective within 1 to 5 years of their introduction for clinical use. This strongly underlines the need for development of new antimalarial therapies possessing long UTLs. The present review explores novel drug targets within the malaria parasite that may be exploited in the search for novel drugs that possess long and UTLs.

Lipoic acid metabolism of Plasmodium--a suitable drug target.

α-Lipoic acid (6,8-thioctic acid; LA) is a vital co-factor of α-ketoacid dehydrogenase complexes and the glycine cleavage system. In recent years it was shown that biosynthesis and salvage of LA in Plasmodium are necessary for the parasites to complete their complex life cycle. LA salvage requires two lipoic acid protein ligases (LplA1 and LplA2). LplA1 is confined to the mitochondrion while LplA2 is located in both the mitochondrion and the apicoplast. LplA1 exclusively uses salvaged LA and lipoylates α-ketoglutarate dehydrogenase, branched chain α-ketoacid dehydrogenase and the H-protein of the glycine cleavage system. LplA2 cannot compensate for the loss of LplA1 function during blood stage development suggesting a specific function for LplA2 that has yet to be elucidated. LA salvage is essential for the intra-erythrocytic and liver stage development of Plasmodium and thus offers great potential for future drug or vaccine development. LA biosynthesis, comprising octanoyl-acyl carrier protein (ACP) : protein N-octanoyltransferase (LipB) and lipoate synthase (LipA), is exclusively found in the apicoplast of Plasmodium where it generates LA de novo from octanoyl-ACP, provided by the type II fatty acid biosynthesis (FAS II) pathway also present in the organelle. LA is the co-factor of the acetyltransferase subunit of the apicoplast located pyruvate dehydrogenase (PDH), which generates acetyl-CoA, feeding into FAS II. LA biosynthesis is not vital for intra-erythrocytic development of Plasmodium, but the deletion of several genes encoding components of FAS II or PDH was detrimental for liver stage development of the parasites indirectly suggesting that the same applies to LA biosynthesis. These data provide strong evidence that LA salvage and biosynthesis are vital for different stages of Plasmodium development and offer potential for drug and vaccine design against malaria.