Structure-based identification of salicylic acid derivatives as malarial threonyl tRNA-synthetase inhibitors.

Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Threonyl t-RNA synthetase (ThrRS) is one of the enzymes involved in this pathway, and it has been validated as an anti-malarial drug target. Here, we present 9 structurally diverse low micromolar Plasmodium falciparum ThrRS inhibitors that were identified using high-throughput virtual screening (HTVS) and were verified in a FRET enzymatic assay. Salicylic acid-based compound (LE = 0.34) was selected as a most perspective hit and was subjected to hit-to-lead optimisation. A total of 146 hit analogues were synthesised or obtained from commercial vendors and were tested. Structure-activity relationship study was supported by the crystal structure of the complex of a salicylic acid analogue with a close homologue of the plasmodium target, E. coli ThrRS (EcThrRS). Despite the availability of structural information, the hit identified via virtual screening remained one of the most potent PfThrRS inhibitors within this series. However, the compounds presented herein provide novel scaffolds for ThrRS inhibitors, which could serve as starting points for further medicinal chemistry projects targeting ThrRSs or structurally similar enzymes.

Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in mainland Tanzania, 2018.

BACKGROUND: The use of artemisinin-based combination therapy (ACT) is recommended by the World Health Organization for the treatment of uncomplicated falciparum malaria. Artemether-lumefantrine (AL) is the most widely adopted first-line ACT for uncomplicated malaria in sub-Saharan Africa (SSA), including mainland Tanzania, where it was introduced in December 2006. The WHO recommends regular assessment to monitor the efficacy of the first-line treatment specifically considering that artemisinin partial resistance was reported in Greater Mekong sub-region and has been confirmed in East Africa (Rwanda and Uganda). The main aim of this study was to assess the efficacy and safety of AL for the treatment of uncomplicated falciparum malaria in mainland Tanzania. METHODS: A single-arm prospective anti-malarial drug efficacy trial was conducted in Kibaha, Mlimba, Mkuzi, and Ujiji (in Pwani, Morogoro, Tanga, and Kigoma regions, respectively) in 2018. The sample size of 88 patients per site was determined based on WHO 2009 standard protocol. Participants were febrile patients (documented axillary temperature ≥ 37.5 °C and/or history of fever during the past 24 h) aged 6 months to 10 years. Patients received a 6-dose AL regimen by weight twice a day for 3 days. Clinical and parasitological parameters were monitored during 28 days of follow-up to evaluate the drug efficacy and safety. RESULTS: A total of 653 children were screened for uncomplicated malaria and 349 (53.7%) were enrolled between April and August 2018. Of the enrolled children, 345 (98.9%) completed the 28 days of follow-up or attained the treatment outcomes. There were no early treatment failures, but recurrent infections were higher in Mkuzi (35.2%) and Ujiji (23%). By Kaplan-Meier analysis of polymerase chain reaction (PCR) uncorrected adequate clinical and parasitological response (ACPR) ranged from 63.4% in Mkuzi to 85.9% in Mlimba, while PCR-corrected ACPR on day 28 varied from 97.6% in Ujiji to 100% in Mlimba. The drug was well tolerated; the commonly reported adverse events were cough, runny nose, and abdominal pain. No serious adverse event was reported. CONCLUSION: This study showed that AL had adequate efficacy and safety for the treatment of uncomplicated falciparum malaria. The high number of recurrent infections were mainly due to new infections, indicating the necessity of utilizing alternative artemisinin-based combinations, such as artesunate amodiaquine, which provide a significantly longer post-treatment prophylactic effect.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing.

BACKGROUND: Understanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing. METHODS: A data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach. RESULTS: A 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68]. CONCLUSIONS: A region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients enrolled at 100 health facilities throughout Tanzania: February to July 2021.

Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.

Plasmodium falciparum genetic diversity and multiplicity of infection based on msp-1, msp-2, glurp and microsatellite genetic markers in sub-Saharan Africa: a systematic review and meta-analysis.

BACKGROUND: In sub-Saharan Africa (SSA), Plasmodium falciparum causes most of the malaria cases. Despite its crucial roles in disease severity and drug resistance, comprehensive data on Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) are sparse in SSA. This study summarizes available information on genetic diversity and MOI, focusing on key markers (msp-1, msp-2, glurp, and microsatellites). The systematic review aimed to evaluate their influence on malaria transmission dynamics and offer insights for enhancing malaria control measures in SSA. METHODS: The review was conducted following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. Two reviewers conducted article screening, assessed the risk of bias (RoB), and performed data abstraction. Meta-analysis was performed using the random-effects model in STATA version 17. RESULTS: The review included 52 articles: 39 cross-sectional studies and 13 Randomized Controlled Trial (RCT)/cohort studies, involving 11,640 genotyped parasite isolates from 23 SSA countries. The overall pooled mean expected heterozygosity was 0.65 (95% CI: 0.51-0.78). Regionally, values varied: East (0.58), Central (0.84), Southern (0.74), and West Africa (0.69). Overall pooled allele frequencies of msp-1 alleles K1, MAD20, and RO33 were 61%, 44%, and 40%, respectively, while msp-2 I/C 3D7 and FC27 alleles were 61% and 55%. Central Africa reported higher frequencies (K1: 74%, MAD20: 51%, RO33: 48%) than East Africa (K1: 46%, MAD20: 42%, RO33: 31%). For msp-2, East Africa had 60% and 55% for I/C 3D7 and FC27 alleles, while West Africa had 62% and 50%, respectively. The pooled allele frequency for glurp was 66%. The overall pooled mean MOI was 2.09 (95% CI: 1.88-2.30), with regional variations: East (2.05), Central (2.37), Southern (2.16), and West Africa (1.96). The overall prevalence of polyclonal Plasmodium falciparum infections was 63% (95% CI: 56-70), with regional prevalences as follows: East (62%), West (61%), Central (65%), and South Africa (71%). CONCLUSION: The study shows substantial regional variation in Plasmodium falciparum parasite genetic diversity and MOI in SSA. These findings suggest a need for malaria control strategies and surveillance efforts considering regional-specific factors underlying Plasmodium falciparum infection.

Antibody response to malaria vaccine candidates in pregnant women with Plasmodium falciparum and Schistosoma haematobium infections.

Malaria in pregnancy has severe consequences for the mother and foetus. Antibody response to specific malaria vaccine candidates (MVC) has been associated with a decreased risk of clinical malaria and its outcomes. We studied Plasmodium falciparum (Pf) and Schistosoma haematobium (Sh) infections and factors that could influence antibody responses to MVC in pregnant women. A total of 337 pregnant women receiving antenatal care (ANC) and 139 for delivery participated in this study. Pf infection was detected by qPCR and Sh infection using urine filtration method. Antibody levels against CSP, AMA-1, GLURP-R0, VAR2CSA and Pfs48/45 MVC were quantified by ELISA. Multivariable linear regression models identified factors associated with the modulation of antibody responses. The prevalence of Pf and Sh infections was 27% and 4% at ANC and 7% and 4% at delivery. Pf infection, residing in Adidome and multigravidae were positively associated with specific IgG response to CSP, AMA-1, GLURP-R0 and VAR2CSA. ITN use and IPTp were negatively associated with specific IgG response to GLURP-R0 and Pfs48/45. There was no association between Sh infection and antibody response to MVC at ANC or delivery. Pf infections in pregnant women were positively associated with antibody response to CSP, GLURP-R0 and AMA-1. Antibody response to GLURP-R0 and Pfs48/45 was low for IPTp and ITN users. This could indicate a lower exposure to Pf infection and low malaria prevalence observed at delivery.

Meet the Authors: Christopher R. Mansfield and Emily R. Derbyshire.

In an interview with Samantha Nelson, a scientific editor of Cell Chemical Biology, the first and corresponding authors of the research article entitled Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome share more about the project and their perspectives on the field.

Ageing of Plasmodium falciparum malaria sporozoites alters their motility, infectivity and reduces immune activation in vitro.

BACKGROUND: Sporozoites (SPZ), the infective form of Plasmodium falciparum malaria, can be inoculated into the human host skin by Anopheline mosquitoes. These SPZ migrate at approximately 1 µm/s to find a blood vessel and travel to the liver where they infect hepatocytes and multiply. In the skin they are still low in number (50-100 SPZ) and vulnerable to immune attack by antibodies and skin macrophages. This is why whole SPZ and SPZ proteins are used as the basis for most malaria vaccines currently deployed and undergoing late clinical testing. Mosquitoes typically inoculate SPZ into a human host between 14 and 25 days after their previous infective blood meal. However, it is unknown whether residing time within the mosquito affects SPZ condition, infectivity or immunogenicity. This study aimed to unravel how the age of P. falciparum SPZ in salivary glands (14, 17, or 20 days post blood meal) affects their infectivity and the ensuing immune responses. METHODS: SPZ numbers, viability by live/dead staining, motility using dedicated sporozoite motility orienting and organizing tool software (SMOOT), and infectivity of HC-04.j7 liver cells at 14, 17 and 20 days after mosquito feeding have been investigated. In vitro co-culture assays with SPZ stimulated monocyte-derived macrophages (MoMɸ) and CD8+ T-cells, analysed by flow cytometry, were used to investigate immune responses. RESULTS: SPZ age did not result in different SPZ numbers or viability. However, a markedly different motility pattern, whereby motility decreased from 89% at day 14 to 80% at day 17 and 71% at day 20 was observed (p ≤ 0.0001). Similarly, infectivity of day 20 SPZ dropped to ~ 50% compared with day 14 SPZ (p = 0.004). MoMɸ were better able to take up day 14 SPZ than day 20 SPZ (from 7.6% to 4.1%, p = 0.03) and displayed an increased expression of pro-inflammatory CD80, IL-6 (p = 0.005), regulatory markers PDL1 (p = 0.02), IL-10 (p = 0.009) and cytokines upon phagocytosis of younger SPZ. Interestingly, co-culture of these cells with CD8+ T-cells revealed a decreased expression of activation marker CD137 and cytokine IFNγ compared to their day 20 counterparts. These findings suggest that older (day 17-20) P. falciparum SPZ are less infectious and have decreased immune regulatory potential. CONCLUSION: Overall, this data is a first step in enhancing the understanding of how mosquito residing time affects P. falciparum SPZ and could impact the understanding of the P. falciparum infectious reservoir and the potency of whole SPZ vaccines.

Disclosure of cinnamic acid/4,9-diaminoacridine conjugates as multi-stage antiplasmodial hits.

4,9-diaminoacridines with reported antiplasmodial activity were coupled to different trans-cinnamic acids, delivering a new series of conjugates inspired by the covalent bitherapy concept. The new compounds were more potent than primaquine against hepatic stages of Plasmodium berghei, although this was accompanied by cytotoxic effects on Huh-7 hepatocytes. Relevantly, the conjugates displayed nanomolar activities against blood stage P. falciparum parasites, with no evidence of hemolytic effects below 100 µM. Moreover, the new compounds were at least 25-fold more potent than primaquine against P. falciparum gametocytes. Thus, the new antiplasmodial hits disclosed herein emerge as valuable templates for the development of multi-stage antiplasmodial drug candidates.

A phase IIa, randomized, double-blind, safety, immunogenicity and efficacy trial of Plasmodium falciparum vaccine antigens merozoite surface protein 1 and RTS,S formulated with AS02 adjuvant in healthy, malaria-naïve adults.

BACKGROUND: To improve the efficacy of Plasmodium falciparum malaria vaccine RTS,S/AS02, we conducted a study in 2001 in healthy, malaria-naïve adults administered RTS,S/AS02 in combination with FMP1, a recombinant merozoite surface-protein-1, C-terminal 42kD fragment. METHODS: A double-blind Phase I/IIa study randomized N = 60 subjects 1:1:1:1 to one of four groups, N = 15/group, to evaluate safety, immunogenicity, and efficacy of intra-deltoid half-doses of RTS,S/AS02 and FMP1/AS02 administered in the contralateral (RTS,S + FMP1-separate) or same (RTS,S + FMP1-same) sites, or FMP1/AS02 alone (FMP1-alone), or RTS,S/AS02 alone (RTS,S-alone) on a 0-, 1-, 3-month schedule. Subjects receiving three doses of vaccine and non-immunized controls (N = 11) were infected with homologous P. falciparum 3D7 sporozoites by Controlled Human Malaria Infection (CHMI). RESULTS: Subjects in all vaccination groups experienced mostly mild or moderate local and general adverse events that resolved within eight days. Anti-circumsporozoite antibody levels were lower when FMP1 and RTS,S were co-administered at the same site (35.0 µg/mL: 95 % CI 20.3-63), versus separate arms (57.4 µg/mL: 95 % CI 32.3-102) or RTS,S alone (62.0 µg/mL: 95 % CI: 37.8-101.8). RTS,S-specific lymphoproliferative responses and ex vivo ELISpot CSP-specific interferon-gamma (IFN-γ) responses were indistinguishable among groups receiving RTS,S/AS02. There was no difference in antibody to FMP1 among groups receiving FMP1/AS02. After CHMI, groups immunized with a RTS,S-containing regimen had âˆ¼ 30 % sterile protection against parasitemia, and equivalent delays in time-to-parasitemia. The FMP1/AS02 alone group showed no sterile immunity or delay in parasitemia. CONCLUSION: Co-administration of RTS,S and FMP1/AS02 reduced anti-RTS,S antibody, but did not affect tolerability, cellular immunity, or efficacy in a stringent CHMI model. Absence of efficacy or delay of patency in the sporozoite challenge model in the FMP1/AS02 group did not rule out efficacy of FMP1/AS02 in an endemic population. However, a Phase IIb trial of FMP1/AS02 in children in malaria-endemic Kenya did not demonstrate efficacy against natural infection. CLINICALTRIALS: gov identifier: NCT01556945.

Numerical analysis and early detection of Plasmodium falciparum using a high performance plasmonic biosensor with an external sensing approach.

The authors propose a surface plasmon resonance (SPR) sensor based on photonic crystal fibers (PCFs) using three hexagonal ring lattices. The sensor can detect biomolecules with maximum wavelength and amplitude sensitivities of 23,000 nm/RIU and 1310.93R I U -1, respectively, in the RI range of 1.32 to 1.42. It can detect infected red blood cells with Plasmodium falciparum for RIs of 1.402, 1.373, 1.395, and 1.383 in various malaria-infected red blood cell stages, including ring phase, trophozoite phase, and schizont phase. Furthermore, the sensor will be able to detect biomolecules such as viruses, proteins, DNA/RNA strands, acetone, ethanol, hexane, isopropanol, hexanol, formic acid, allyl cyanide, and others in its range. With these impressive results and identification capacity, the proposed sensor would benefit the biomaterial field and be appropriate for the early identification of malaria disease.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine and prevalence of molecular markers of anti-malarial drug resistance in children in Togo in 2021.

BACKGROUND: Artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the currently recommended first- and second-line therapies for uncomplicated Plasmodium falciparum infections in Togo. This study assessed the efficacy of these combinations, the proportion of Day3-positive patients (D3 +), the proportion of molecular markers associated with P. falciparum resistance to anti-malarial drugs, and the variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). METHODS: A single arm prospective study evaluating the efficacy of AL and DP was conducted at two sites (Kouvé and Anié) from September 2021 to January 2022. Eligible children were enrolled, randomly assigned to treatment at each site and followed up for 42 days after treatment initiation. The primary endpoint was polymerase chain reaction (PCR) adjusted adequate clinical and parasitological response (ACPR). At day 0, samples were analysed for mutations in the Pfkelch13, Pfcrt, Pfmdr-1, dhfr, dhps, and deletions in the hrp2/hrp3 genes. RESULTS: A total of 179 and 178 children were included in the AL and DP groups, respectively. After PCR correction, cure rates of patients treated with AL were 97.5% (91.4-99.7) at day 28 in Kouvé and 98.6% (92.4-100) in Anié, whereas 96.4% (CI 95%: 89.1-98.8) and 97.3% (CI 95%: 89.5-99.3) were observed at day 42 in Kouvé and Anié, respectively. The cure rates of patients treated with DP at day 42 were 98.9% (CI 95%: 92.1-99.8) in Kouvé and 100% in Anié. The proportion of patients with parasites on day 3 (D3 +) was 8.5% in AL and 2.6% in DP groups in Anié and 4.3% in AL and 2.1% DP groups in Kouvé. Of the 357 day 0 samples, 99.2% carried the Pfkelch13 wild-type allele. Two isolates carried nonsynonymous mutations not known to be associated with artemisinin partial resistance (ART-R) (A578S and A557S). Most samples carried the Pfcrt wild-type allele (97.2%). The most common Pfmdr-1 allele was the single mutant 184F (75.6%). Among dhfr/dhps mutations, the quintuple mutant haplotype N51I/C59R/S108N + 437G/540E, which is responsible for SP treatment failure in adults and children, was not detected. Single deletions in hrp2 and hrp3 genes were detected in 1/357 (0.3%) and 1/357 (0.3%), respectively. Dual hrp2/hrp3 deletions, which could affect the performances of HRP2-based RDTs, were not observed. CONCLUSION: The results of this study confirm that the AL and DP treatments are highly effective. The absence of the validated Pfkelch13 mutants in the study areas suggests the absence of ART -R, although a significant proportion of D3 + cases were found. The absence of dhfr/dhps quintuple or sextuple mutants (quintuple + 581G) supports the continued use of SP for IPTp during pregnancy and in combination with amodiaquine for seasonal malaria chemoprevention. TRIAL REGISTRATION: ACTRN12623000344695.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in mainland Tanzania, 2019.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been a major contributor to the substantial reductions in global malaria morbidity and mortality over the last decade. In Tanzania, artemether-lumefantrine (AL) was introduced as the first-line treatment for uncomplicated Plasmodium falciparum malaria in 2006. The World Health Organization (WHO) recommends regular assessment and monitoring of the efficacy of the first-line treatment, specifically considering that artemisinin resistance has been confirmed in the Greater Mekong sub-region. This study's main aim was to assess the efficacy and safety of AL for treating uncomplicated P. falciparum malaria in Tanzania. METHODS: This was a single-arm prospective antimalarial drug efficacy trial conducted in four of the eight National Malaria Control Programme (NMCP) sentinel sites in 2019. The trial was carried out in outpatient health facilities in Karume-Mwanza region, Ipinda-Mbeya region, Simbo-Tabora region, and Nagaga-Mtwara region. Children aged six months to 10 years with microscopy confirmed uncomplicated P. falciparum malaria who met the inclusion criteria were recruited based on the WHO protocol. The children received AL (a 6-dose regimen of AL twice daily for three days). Clinical and parasitological parameters were monitored during follow-up over 28 days to evaluate drug efficacy. RESULTS: A total of 628 children were screened for uncomplicated malaria, and 349 (55.6%) were enrolled between May and September 2019. Of the enrolled children, 343 (98.3%) completed the 28-day follow-up or attained the treatment outcomes. There were no early treatment failures; recurrent infections during follow-up were common at two sites (Karume 29.5%; Simbo 18.2%). PCR-corrected adequate clinical and parasitological response (ACPR) by survival analysis to AL on day 28 of follow-up varied from 97.7% at Karume to 100% at Ipinda and Nagaga sites. The commonly reported adverse events were cough, skin pallor, and abdominal pain. The drug was well tolerated, and no serious adverse event was reported. CONCLUSION: This study showed that AL had adequate efficacy and safety for the treatment of uncomplicated falciparum malaria in Tanzania in 2019. The high recurrent infections were mainly due to new infections, highlighting the potential role of introducing alternative artemisinin-based combinations that offer improved post-treatment prophylaxis, such as artesunate-amodiaquine (ASAQ).

Next Generation Chemiluminescent Probes for Antimalarial Drug Discovery.

Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark ß-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (ß-D-galactoside) by a transgenic ß-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.

Let it glow: genetically encoded fluorescent reporters in Plasmodium.

The use of fluorescent proteins (FPs) in Plasmodium parasites has been key to understand the biology of this obligate intracellular protozoon. FPs like the green fluorescent protein (GFP) enabled to explore protein localization, promoter activity as well as dynamic processes like protein export and endocytosis. Furthermore, FP biosensors have provided detailed information on physiological parameters at the subcellular level, and fluorescent reporter lines greatly extended the malariology toolbox. Still, in order to achieve optimal results, it is crucial to know exactly the properties of the FP of choice and the genetic scenario in which it will be used. This review highlights advantages and disadvantages of available landing sites and promoters that have been successfully applied for the ectopic expression of FPs in Plasmodium berghei and Plasmodium falciparum. Furthermore, the properties of newly developed FPs beyond DsRed and EGFP, in the visualization of cells and cellular structures as well as in the sensing of small molecules are discussed.

Widespread pfhrp2/3 deletions and HRP2-based false-negative results in southern Ethiopia.

BACKGROUND: Rapid diagnostic tests (RDTs) play a significant role in expanding case management in peripheral healthcare systems. Histidine-rich protein-2 (HRP2) antigen detection RDTs are predominantly used to diagnose Plasmodium falciparum infection. However, the evolution and spread of P. falciparum parasite strains with deleted hrp2/3 genes, causing false-negative results, have been reported. This study assessed the diagnostic performance of HRP2-detecting RDTs for P. falciparum cases and the prevalence of pfhrp2/3 deletions among symptomatic patients seeking malaria diagnosis at selected health facilities in southern Ethiopia. METHODS: A multi-health facilities-based cross-sectional study was conducted on self-presenting febrile patients seeking treatment in southern Ethiopia from July to September 2022. A purposive sampling strategy was used to enroll patients with microscopically confirmed P. falciparum infections. A capillary blood sample was obtained to prepare a blood film for microscopy and a RDT using the SD Bioline™ Malaria Pf/Pv Test. Dried blood spot samples were collected for further molecular analysis. DNA was extracted using gene aid kits and amplification was performed using nested PCR assay. Exon 2 of hrp2 and hrp3, which are the main protein-coding regions, was used to confirm its deletion. The diagnostic performance of RDT was evaluated using PCR as the gold standard test for P. falciparum infections. RESULTS: Of 279 P. falciparum PCR-confirmed samples, 249 (89.2%) had successful msp-2 amplification, which was then genotyped for hrp2/3 gene deletions. The study revealed that pfhrp2/3 deletions were common in all health centres, and it was estimated that 144 patients (57.8%) across all health facilities had pfhrp2/3 deletions, leading to false-negative PfHRP2 RDT results. Deletions spanning exon 2 of hrp2, exon 2 of hrp3, and double deletions (hrp2/3) accounted for 68 (27.3%), 76 (30.5%), and 33 (13.2%) of cases, respectively. The study findings revealed the prevalence of P. falciparum parasites lacking a single pfhrp2-/3-gene and that both genes varied across the study sites. This study also showed that the sensitivity of the SD Bioline PfHRP2-RDT test was 76.5% when PCR was used as the reference test. CONCLUSION: This study confirmed the existence of widespread pfhrp2/3- gene deletions, and their magnitude exceeded the WHO-recommended threshold (> 5%). False-negative RDT results resulting from deletions in Pfhrp2/3- affect a country's attempts at malaria control and elimination. Therefore, the adoption of non-HRP2-based RDTs as an alternative measure is required to avoid the consequences associated with the continued use of HRP-2-based RDTs, in the study area in particular and in Ethiopia in general.

Solution structure and pressure response of thioredoxin-1 of Plasmodium falciparum.

We present here the solution structures of the protein thioredoxin-1 from Plasmodium falciparum (PfTrx-1), in its reduced and oxidized forms. They were determined by high-resolution NMR spectroscopy at 293 K on uniformly 13C-, 15N-enriched, matched samples allowing to identification of even small structural differences. PfTrx-1 shows an α/ß-fold with a mixed five-stranded ß-sheet that is sandwiched between 4 helices in a ß1 α1 ß2 α2 ß3 α3 ß4 ß5 α4 topology. The redox process of the CGPC motif leads to significant structural changes accompanied by larger chemical shift changes from residue Phe25 to Ile36, Thr70 to Thr74, and Leu88 to Asn91. By high-field high-pressure NMR spectroscopy, rare conformational states can be identified that potentially are functionally important and can be used for targeted drug development. We performed these experiments in the pressure range from 0.1 MPa to 200 MPa. The mean combined, random-coil corrected B1* values of reduced and oxidized thioredoxin are quite similar with -0.145 and -0.114 ppm GPa-1, respectively. The mean combined, random-coil corrected B2* values in the reduced and oxidized form are 0.179 and 0.119 ppm GPa-2, respectively. The mean ratios of the pressure coefficients B2/B1 are -0.484 and -0.831 GPa-1 in the reduced and oxidized form respectively. They differ at some points in the structure after the formation of the disulfide bond between C30 and C33. The thermodynamical description of the pressure dependence of chemical shifts requires the assumption of at least three coexisting conformational states of PfTrx-1. These three conformational states were identified in the reduced as well as in the oxidized form of the protein, therefore, they represent sub-states of the two main oxidation states of PfTrx-1.

A Pfs48/45-based vaccine to block Plasmodium falciparum transmission: phase 1, open-label, clinical trial.

BACKGROUND: The stalling global progress in malaria control highlights the need for novel tools for malaria elimination, including transmission-blocking vaccines. Transmission-blocking vaccines aim to induce human antibodies that block parasite development in the mosquito and mosquitoes becoming infectious. The Pfs48/45 protein is a leading Plasmodium falciparum transmission-blocking vaccine candidate. The R0.6C fusion protein, consisting of Pfs48/45 domain 3 (6C) and the N-terminal region of P. falciparum glutamate-rich protein (R0), has previously been produced in Lactococcus lactis and elicited functional antibodies in rodents. Here, we assess the safety and transmission-reducing efficacy of R0.6C adsorbed to aluminium hydroxide with and without Matrix-M™ adjuvant in humans. METHODS: In this first-in-human, open-label clinical trial, malaria-naïve adults, aged 18-55 years, were recruited at the Radboudumc in Nijmegen, the Netherlands. Participants received four intramuscular vaccinations on days 0, 28, 56 and 168 with either 30 µg or 100 µg of R0.6C and were randomised for the allocation of one of the two different adjuvant combinations: aluminium hydroxide alone, or aluminium hydroxide combined with Matrix-M1™ adjuvant. Adverse events were recorded from inclusion until 84 days after the fourth vaccination. Anti-R0.6C and anti-6C IgG titres were measured by enzyme-linked immunosorbent assay. Transmission-reducing activity of participants' serum and purified vaccine-specific immunoglobulin G was assessed by standard membrane feeding assays using laboratory-reared Anopheles stephensi mosquitoes and cultured P. falciparum gametocytes. RESULTS: Thirty-one participants completed four vaccinations and were included in the analysis. Administration of all doses was safe and well-tolerated, with one related grade 3 adverse event (transient fever) and no serious adverse events occurring. Anti-R0.6C and anti-6C IgG titres were similar between the 30 and 100 µg R0.6C arms, but higher in Matrix-M1™ arms. Neat participant sera did not induce significant transmission-reducing activity in mosquito feeding experiments, but concentrated vaccine-specific IgGs purified from sera collected two weeks after the fourth vaccination achieved up to 99% transmission-reducing activity. CONCLUSIONS: R0.6C/aluminium hydroxide with or without Matrix-M1™ is safe, immunogenic and induces functional Pfs48/45-specific transmission-blocking antibodies, albeit at insufficient serum concentrations to result in transmission reduction by neat serum. Future work should focus on identifying alternative vaccine formulations or regimens that enhance functional antibody responses. TRIAL REGISTRATION: The trial is registered with ClinicalTrials.gov under identifier NCT04862416.

MYST regulates DNA repair and forms a NuA4-like complex in the malaria parasite Plasmodium falciparum.

Histone lysine acetyltransferase MYST-associated NuA4 complex is conserved from yeast to humans and plays key roles in cell cycle regulation, gene transcription, and DNA replication/repair. Here, we identified a Plasmodium falciparum MYST-associated complex, PfNuA4, which contains 11 of the 13 conserved NuA4 subunits. Reciprocal pulldowns using PfEAF2, a shared component between the NuA4 and SWR1 complexes, not only confirmed the PfNuA4 complex but also identified the PfSWR1 complex, a histone remodeling complex, although their identities are low compared to the homologs in yeast or humans. Notably, both H2A.Z/H2B.Z were associated with the PfSWR1 complex, indicating that this complex is involved in the deposition of H2A.Z/H2B.Z, the variant histone pair that is enriched in the activated promoters. Overexpression of PfMYST resulted in earlier expression of genes involved in cell cycle regulation, DNA replication, and merozoite invasion, and upregulation of the genes related to antigenic variation and DNA repair. Consistently, PfMYST overexpression led to high basal phosphorylated PfH2A (γ-PfH2A), the mark of DNA double-strand breaks, and conferred protection against genotoxic agent methyl methanesulfonate (MMS), X-rays, and artemisinin, the first-line antimalarial drug. In contrast, the knockdown of PfMYST caused a delayed parasite recovery upon MMS treatment. MMS induced the gradual disappearance of PfMYST in the cytoplasm and concomitant accumulation of PfMYST in the nucleus, suggesting cytoplasm-nucleus shuttling of PfMYST. Meanwhile, PfMYST colocalized with the γ-PfH2A, indicating PfMYST was recruited to the DNA damage sites. Collectively, PfMYST plays critical roles in cell cycle regulation, gene transcription, and DNA replication/DNA repair in this low-branching parasitic protist.IMPORTANCEUnderstanding gene regulation and DNA repair in malaria parasites is critical for identifying targets for antimalarials. This study found PfNuA4, a PfMYST-associated, histone modifier complex, and PfSWR1, a chromatin remodeling complex in malaria parasite Plasmodium falciparum. These complexes are divergent due to the low identities compared to their homologs from yeast and humans. Furthermore, overexpression of PfMYST resulted in substantial transcriptomic changes, indicating that PfMYST is involved in regulating the cell cycle, antigenic variation, and DNA replication/repair. Consistently, PfMYST was found to protect against DNA damage caused by the genotoxic agent methyl methanesulfonate, X-rays, and artemisinin, the first-line antimalarial drug. Additionally, DNA damage led to the relocation of cytoplasmic PfMYST to the nucleus and colocalization of PfMYST with γ-PfH2A, the mark of DNA damage. In summary, this study demonstrated that the PfMYST complex has critical functions in regulating cell cycle, antigenic variation, and DNA replication/DNA repair in P. falciparum.