Detection of novel Plasmodium falciparum coronin gene mutations in a recrudescent ACT-treated patient in South-Western Nigeria.

Background: Routine surveillance for antimalarial drug resistance is critical to sustaining the efficacy of artemisinin-based Combination Therapies (ACTs). Plasmodium falciparum kelch-13 (Pfkelch-13) and non-Pfkelch-13 artemisinin (ART) resistance-associated mutations are uncommon in Africa. We investigated polymorphisms in Plasmodium falciparum actin-binding protein (Pfcoronin) associated with in vivo reduced sensitivity to ART in Nigeria. Methods: Fifty-two P. falciparum malaria subjects who met the inclusion criteria were followed up in a 28-day therapeutic efficacy study of artemether-lumefantrine in Lagos, Nigeria. Parasite detection was done by microscopy and molecular diagnostic approaches involving PCR amplification of genes for Pf18S rRNA, varATS, telomere-associated repetitive elements-2 (TARE-2). Pfcoronin and Pfkelch-13 genes were sequenced bi-directionally while clonality of infections was determined using 12 neutral P. falciparum microsatellite loci and msp2 analyses. Antimalarial drugs (sulfadoxine-pyrimethamine, amodiaquine, chloroquine and some quinolones) resistance variants (DHFR_51, DHFR_59, DHFR_108, DHFR_164, MDR1_86, MDR1_184, DHPS_581 and DHPS_613) were genotyped by high-resolution melting (HRM) analysis. Results: A total of 7 (26.92%) cases were identified either as early treatment failure, late parasitological failure or late clinical failure. Of the four post-treatment infections identified as recrudescence by msp2 genotypes, only one was classified as recrudescence by multilocus microsatellites genotyping. Microsatellite analysis revealed no significant difference in the mean allelic diversity, He, (P = 0.19, Mann-Whitney test). Allele sizes and frequency per locus implicated one isolate. Genetic analysis of this isolate identified two new Pfcoronin SNVs (I68G and L173F) in addition to the P76S earlier reported. Linkage-Disequilibrium as a standardized association index, IAS, between multiple P. falciparum loci revealed significant LD (IAS = 0.2865, P=0.02, Monte-Carlo simulation) around the neutral microsatellite loci. The pfdhfr/pfdhps/pfmdr1 drug resistance-associated haplotypes combinations, (108T/N/51I/164L/59R/581G/86Y/184F), were observed in two samples. Conclusion: Pfcoronin mutations identified in this study, with potential to impact parasite clearance, may guide investigations on emerging ART tolerance in Nigeria, and West African endemic countries.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

Molecular markers of artemisinin resistance during falciparum malaria elimination in Eastern Myanmar.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013-2019) was characterized. METHODS: Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13-a molecular marker of artemisinin resistance. RESULT: The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p < 0.001). In the MDA villages there was no significant change in the K13 proportions before and after MDA. The distribution of different K13 mutations changed substantially; F446I and P441L mutations increased in both MDA and non-MDA villages, while most other K13 mutations decreased. The proportion of C580Y mutations fell from 9.2% (43/467) before MDA to 2.3% (19/813) after MDA (p < 0.001). Similar changes occurred in the 487 villages where MDA was not conducted. CONCLUSION: The malaria elimination program in Kayin state, eastern Myanmar, led to a substantial reduction in falciparum malaria. Despite the intense use of artemisinin-based combination therapies, both in treatment and MDA, this did not select for artemisinin resistance.

A simple, field-applicable method to increase the infectivity of wild isolates of Plasmodium falciparum to mosquito vectors.

BACKGROUND: The direct membrane feeding assay (DMFA), whereby gametocyte-infected blood is collected from human donors and from which mosquitoes feed through a membrane, is proving essential for assessing parameters influencing Plasmodium transmission potential in endemic countries. The success of DMFAs is closely tied to gametocyte density in the blood, with relatively high gametocytaemia ensuring optimal infection levels in mosquitoes. As transmission intensity declines with control efforts, the occurrence of asymptomatic individuals with low gametocyte densities, who can significantly contribute to the infectious reservoir, is increasing. This poses a limitation to studies relying on the experimental infection of large numbers of mosquitoes with natural isolates of Plasmodium. A simple, field-applicable method is presented for improving parasite infectivity by concentrating Plasmodium falciparum gametocytes. METHODS: Anopheles gambiae received one of the following 5 blood treatments through DMFA: (i) whole blood (WB) samples from naturally-infected donors; (ii) donor blood whose plasma was replaced with the same volume of Plasmodium-naive AB + serum (1:1 control); (iii) plasma replaced with a volume of malaria-naïve AB + serum equivalent to half (1:1/2), or to a quarter (1:1/4), of the initial plasma volume; and (v) donor blood whose plasma was fully removed (RBC). The experiment was repeated 4 times using 4 distinct wild parasite isolates. Seven days post-infection, a total of 1,095 midguts were examined for oocyst presence. RESULTS: Substituting plasma with reduced amounts (1:1/2 and 1:1/4) of Plasmodium-naive AB + serum led to a 31% and 17% increase of the mosquito infection rate and to a 85% and 308% increase in infection intensity compared to the 1:1 control, respectively. The full removal of plasma (RBC) reduced the infection rate by 58% and the intensity by 64% compared to the 1:1 control. Reducing serum volumes (1:1/2; 1:1/4 and RBC) had no impact on mosquito feeding rate and survival when compared to the 1:1 control. CONCLUSIONS: Concentrating gametocytic blood by replacing natural plasma by lower amount of naive serum can enhance the success of mosquito infection. In an area with low gametocyte density, this simple and practical method of parasite concentration can facilitate studies on human-to-mosquito transmission such as the evaluation of transmission-blocking interventions.

Comparative evaluation of the diagnostic accuracies of four different malaria rapid diagnostic test kits available in Ghana.

Malaria rapid diagnostic test (mRDT) kit is one of the techniques for diagnosing malaria. Due to its inherent advantages over the microscopy technique, several brands of the kit have flooded malaria endemic countries, without prior in-country evaluation. Two of such mRDT kits are Oscar (India) and Standard Q (Korea Republic). In this study, the performance of Oscar and Standard Q mRDT kits were compared to First Response (India) and CareStart (USA) mRDTs, which have been evaluated and deployed for use approved by the Ministry of Health (MOH). In this comparative study, whole blood samples were collected from patients suspected of malaria. Plasmodium falciparum was detected in each sample using nested polymerase chain reaction (nPCR), microscopy and the four mRDTs. The sensitivities, specificities, accuracies, positive and negative predictive values and accuracies of the mRDTs were determined using nPCR as a reference technique. Kappa statistic was used to determine the level of agreement among the techniques. Two hundred (200) blood samples were analyzed in this study. The overall detection rates of P. falciparum by microscopy, First Response, CareStart, Oscar-PfHRP2, Standard Q mRDT kits and nPCR were 31.5%, 34.5%, 33.5%, 32%, 31% and 43% (x2 = 6.1, p = 0.046), respectively. The accuracies of CareStart and First Response were comparable (90.5% vs. 89.5%). Further, comparing their sensitivities, Oscar-PfHRP2 was 74.4% (95% confidence interval (CI): 63.9-83.2) while that of Standard Q was 72.1% (95% CI: 61.4-81.2), with comparable accuracies (Oscar-PfHRP2-89% and Standard Q -88%). Apart from First Response that was 98.3% specific, the others were 100% specific. Kappa test revealed perfect diagnostic agreement (κ = 0.90-0.98) among the four mRDTs. That notwithstanding, Oscar-PfHRP2 agreed better with CareStart (κ = 0.94) and First Response (κ = 0.92) compared to the agreement between Standard Q and, CareStart (κ = 0.92) and First Response (κ = 0.90). Taken together, the diagnostic performance of the four mRDT kits were statistically similar. That notwithstanding, new mRDT kits should be evaluated prior to deployment for use.

Competitive Inhibition of Okanin against Plasmodium falciparum Tyrosyl-tRNA Synthetase.

Malaria is a severe disease that presents a significant threat to human health. As resistance to current drugs continues to increase, there is an urgent need for new antimalarial medications. Aminoacyl-tRNA synthetases (aaRSs) represent promising targets for drug development. In this study, we identified Plasmodium falciparum tyrosyl-tRNA synthetase (PfTyrRS) as a potential target for antimalarial drug development through a comparative analysis of the amino acid sequences and three-dimensional structures of human and plasmodium TyrRS, with particular emphasis on differences in key amino acids at the aminoacylation site. A total of 2141 bioactive compounds were screened using a high-throughput thermal shift assay (TSA). Okanin, known as an inhibitor of LPS-induced TLR4 expression, exhibited potent inhibitory activity against PfTyrRS, while showing limited inhibition of human TyrRS. Furthermore, bio-layer interferometry (BLI) confirmed the high affinity of okanin for PfTyrRS. Molecular dynamics (MD) simulations highlighted the stable conformation of okanin within PfTyrRS and its sustained binding to the enzyme. A molecular docking analysis revealed that okanin binds to both the tyrosine and partial ATP binding sites of the enzyme, preventing substrate binding. In addition, the compound inhibited the production of Plasmodium falciparum in the blood stage and had little cytotoxicity. Thus, okanin is a promising lead compound for the treatment of malaria caused by P. falciparum.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

A single cell atlas of sexual development in Plasmodium falciparum.

The developmental decision made by malaria parasites to become sexual underlies all malaria transmission. Here, we describe a rich atlas of short- and long-read single-cell transcriptomes of over 37,000 Plasmodium falciparum cells across intraerythrocytic asexual and sexual development. We used the atlas to explore transcriptional modules and exon usage along sexual development and expanded it to include malaria parasites collected from four Malian individuals naturally infected with multiple P. falciparum strains. We investigated genotypic and transcriptional heterogeneity within and among these wild strains at the single-cell level, finding differential expression between different strains even within the same host. These data are a key addition to the Malaria Cell Atlas interactive data resource, enabling a deeper understanding of the biology and diversity of transmission stages.

Plasmodium falciparum alters the trophoblastic barrier and stroma villi organization of human placental villi explants.

BACKGROUND: The sequestration of Plasmodium falciparum infected erythrocytes in the placenta, and the resulting inflammatory response affects maternal and child health. Despite existing information, little is known about the direct impact of P. falciparum on the placental barrier formed by trophoblast and villous stroma. This study aimed to assess placental tissue damage caused by P. falciparum in human placental explants (HPEs). METHODS: HPEs from chorionic villi obtained of human term placentas (n = 9) from normal pregnancies were exposed to P. falciparum-infected erythrocytes (IE) for 24 h. HPEs were embedded in paraffin blocks and used to study tissue damage through histopathological and histochemical analysis and apoptosis using TUNEL staining. Culture supernatants were collected to measure cytokine and angiogenic factors and to determine LDH activity as a marker of cytotoxicity. A subset of archived human term placenta paraffin-embedded blocks from pregnant women with malaria were used to confirm ex vivo findings. RESULTS: Plasmodium falciparum-IE significantly damages the trophoblast layer and the villous stroma of the chorionic villi. The increased LDH activity and pathological findings such as syncytial knots, fibrin deposits, infarction, trophoblast detachment, and collagen disorganization supported these findings. The specific damage to the trophoblast and the thickening of the subjacent basal lamina were more pronounced in the ex vivo infection. In contrast, apoptosis was higher in the in vivo infection. This disparity could be attributed to the duration of exposure to the infection, which significantly varied between individuals naturally exposed over time and the 24-h exposure in the ex vivo HPE model. CONCLUSION: Exposure to P. falciparum-IE induces a detachment of the syncytiotrophoblast, disorganization of the stroma villi, and an increase in apoptosis, alterations that may be associated with adverse results such as intrauterine growth restriction and low birth weight.

Alteration of prothrombin time in Plasmodium falciparum and Plasmodium vivax infections with different levels of severity: a systematic review and meta-analysis.

Malaria infection leads to hematological abnormalities, including deranged prothrombin time (PT). Given the inconsistent findings regarding PT in malaria across different severities and between Plasmodium falciparum and P. vivax, this study aimed to synthesize available evidence on PT variations in clinical malaria. A systematic literature search was performed in PubMed, Embase, Scopus, Ovid, and Medline from 27 November 2021 to 2 March 2023 to obtain studies documenting PT in malaria. Study quality was evaluated using the Joanna Briggs Institute checklist, with data synthesized through both qualitative and quantitative methods, including meta-regression and subgroup analyses, to explore heterogeneity and publication bias. From 2767 articles, 21 studies were included. Most studies reported prolonged or increased PT in malaria patients compared to controls, a finding substantiated by the meta-analysis (P < 0.01, Mean difference: 8.86 s, 95% CI 5.32-12.40 s, I2: 87.88%, 4 studies). Severe malaria cases also showed significantly higher PT than non-severe ones (P = 0.03, Hedges's g: 1.65, 95% CI 0.20-3.10, I2: 97.91%, 7 studies). No significant PT difference was observed between P. falciparum and P. vivax infections (P = 0.88, Mean difference: 0.06, 95% CI - 0.691-0.8, I2: 65.09%, 2 studies). The relationship between PT and malaria-related mortality remains unclear, underscoring the need for further studies. PT is typically prolonged or increased in malaria, particularly in severe cases, with no notable difference between P. falciparum and P. vivax infections. The inconsistency in PT findings between fatal and non-fatal cases highlights a gap in current understanding, emphasizing the need for future studies to inform therapeutic strategies.

Prevalence of malaria and associated risk factors among household members in South Ethiopia: a multi-site cross-sectional study.

BACKGROUND: Despite continuous prevention and control strategies in place, malaria remains a major public health problem in sub-Saharan Africa including Ethiopia. Moreover, prevalence of malaria differs in different geographical settings and epidemiological data were inadequate to assure disease status in the study area. This study was aimed to determine the prevalence of malaria and associated risk factors in selected rural kebeles in South Ethiopia. METHODS: A community-based cross-sectional study was conducted between February to June 2019 in eight malaria-endemic kebeles situated in four zones in South Ethiopia. Mult-stage sampling techniques were employed to select the study zones, districts, kebeles and households. Blood sample were collected from 1674 participants in 345 households by finger prick and smears were examined by microscopy. Sociodemographic data as well as risk factors for Plasmodium infection were collected using questionnaires. Bivariate and multivariate logistic regressions were used to analyse the data. RESULTS: The overall prevalence of malaria in the study localities was 4.5% (76/1674). The prevalence was varied among the study localities with high prevalence in Bashilo (14.6%; 33/226) followed by Mehal Korga (12.1%; 26/214). Plasmodium falciparum was the dominant parasite accounted for 65.8% (50/76), while Plasmodium vivax accounted 18.4% (14/76). Co-infection of P. falciparum and P. vivax was 15.8% (12/76). Among the three age groups prevalence was 7.8% (27/346) in age less than 5 years and 7.5% (40/531) in 5-14 years. The age groups > 14years were less likely infected with Plasmodium parasite (AOR = 0.14, 95% CI 0.02-0.82) than under five children. Non-febrile individuals 1638 (97.8%) were more likely to had Plasmodium infection (AOR = 28.4, 95% CI 011.4-70.6) than febrile 36 (2.2%). Individuals living proximity to mosquito breeding sites have higher Plasmodium infection (AOR = 6.17, 95% CI 2.66-14.3) than those at distant of breeding sites. CONCLUSIONS: Malaria remains a public health problem in the study localities. Thus, malaria prevention and control strategies targeting children, non-febrile cases and individuals living proximity to breeding sites are crucial to reduce malaria related morbidity and mortality.

Sub-microscopic Plasmodium falciparum infections and multiple drug resistant single nucleotide polymorphic alleles in pregnant women from southwestern Nigeria.

OBJECTIVES: The study evaluated sub-microscopic malaria infections in pregnancy using two malaria Rapid Diagnostic Tests (mRDTs), microscopy and RT-PCR and characterized Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthase (Pfdhps) drug resistant markers in positive samples. METHODS: This was a cross sectional survey of 121 pregnant women. Participants were finger pricked, blood drops were collected for rapid diagnosis with P. falciparum histidine-rich protein 11 rapid diagnostic test kit and the ultra-sensitive Alere Pf malaria RDT, Blood smears for microscopy and dried blood spots on Whatman filter paper for molecular analysis were made. Real time PCR targeting the var acidic terminal sequence (varATS) gene of P. falciparum was carried out on a CFX 96 real time system thermocycler (BioRad) in discriminating malaria infections. For each run, laboratory strain of P. falciparum 3D7 and nuclease free water were used as positive and negative controls respectively. Additionally, High resolution melt analyses was employed for genotyping of the different drug resistance markers. RESULTS: Out of one hundred and twenty-one pregnant women sampled, the SD Bioline™ Malaria Ag P.f HRP2-based malaria rapid diagnostic test (mRDT) detected eight (0.06%) cases, the ultra-sensitive Alere™ malaria Ag P.f rapid diagnostic test mRDT had similar outcome in the same samples as detected by the HRP2-based mRDT. Microscopy and RT-PCR confirmed four out of the eight infections detected by both rapid diagnostic tests as true positive and RT-PCR further detected three false negative samples by the two mRDTs providing a sub-microscopic malaria prevalence of 3.3%. Single nucleotide polymorphism in Pfdhps gene associated with sulphadoxine resistance revealed the presence of S613 mutant genotypes in three of the seven positive isolates and isolates with mixed wild/mutant genotype at codon A613S. Furthermore, four mixed genotypes at the A581G codon were also recorded while the other Pfdhps codons (A436G, A437G and K540E) showed the presence of wild type alleles. In the Pfdhfr gene, there were mutations in 28.6%, 28.6%, and 85.7% at the I51, R59 and N108 codons respectively. Mixed wild and mutant type genotypes were also observed in 28.6% each of the N51I, and C59R codons. For the Pfcrt, two haplotypes CVMNK and CVIET were observed. The SVMNT was altogether absent. Triple mutant CVIET 1(14.3%) and triple mutant + wild genotype CVIET + CVMNK 1(14.3%) were observed. The Pfmdr1 haplotypes were single mutants YYND 1(14.3%); NFND 1(14.3%) and double mutants YFND 4(57.1%); YYDD 1(14.3%).

Persistent and multiclonal malaria parasite dynamics despite extended artemether-lumefantrine treatment in children.

Standard diagnostics used in longitudinal antimalarial studies are unable to characterize the complexity of submicroscopic parasite dynamics, particularly in high transmission settings. We use molecular markers and amplicon sequencing to characterize post-treatment stage-specific malaria parasite dynamics during a 42 day randomized trial of 3- versus 5 day artemether-lumefantrine in 303 children with and without HIV (ClinicalTrials.gov number NCT03453840). The prevalence of parasite-derived 18S rRNA is >70% in children throughout follow-up, and the ring-stage marker SBP1 is detectable in over 15% of children on day 14 despite effective treatment. We find that the extended regimen significantly lowers the risk of recurrent ring-stage parasitemia compared to the standard 3 day regimen, and that higher day 7 lumefantrine concentrations decrease the probability of ring-stage parasites in the early post-treatment period. Longitudinal amplicon sequencing reveals remarkably dynamic patterns of multiclonal infections that include new and persistent clones in both the early post-treatment and later time periods. Our data indicate that post-treatment parasite dynamics are highly complex despite efficacious therapy, findings that will inform strategies to optimize regimens in the face of emerging partial artemisinin resistance in Africa.

Association between telomere length and Plasmodium falciparum malaria endemicity in sub-Saharan Africans.

Leukocyte telomere length (LTL) varies significantly across human populations, with individuals of African ancestry having longer LTL than non-Africans. However, the genetic and environmental drivers of LTL variation in Africans remain largely unknown. We report here on the relationship between LTL, genetics, and a variety of environmental and climatic factors in ethnically diverse African adults (n = 1,818) originating from Botswana, Tanzania, Ethiopia, and Cameroon. We observe significant variation in LTL among populations, finding that the San hunter-gatherers from Botswana have the longest leukocyte telomeres and that the Fulani pastoralists from Cameroon have the shortest telomeres. Genetic factors explain ∼50% of LTL variation among individuals. Moreover, we observe a significant negative association between Plasmodium falciparum malaria endemicity and LTL while adjusting for age, sex, and genetics. Within Africa, adults from populations indigenous to areas with high malaria exposure have shorter LTL than those in populations indigenous to areas with low malaria exposure. Finally, we explore to what degree the genetic architecture underlying LTL in Africa covaries with malaria exposure.

Expansion of artemisinin partial resistance mutations and lack of histidine rich protein-2 and -3 deletions in Plasmodium falciparum infections from Rukara, Rwanda.

BACKGROUND: Emerging artemisinin partial resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (k13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. k13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015, but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, an assessment was conducted to evaluate recent k13-561H prevalence changes, as well as other key mutations. Prevalence of hrp2/3 deletions was also assessed. METHODS: Samples collected in Rukara in 2021 were genotyped for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR. RESULTS: Clinically validated k13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of k13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other anti-malarials were variable, with high levels of multidrug resistance 1 (mdr1) N86 (95.5%) associated with lumefantrine decreased susceptibility and dihydrofolate reductase (dhfr) 164L (24.7%) associated with a high level of antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (crt) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found. CONCLUSIONS: Increasing prevalence of artemisinin partial resistance due to k13-561H and the rapid expansion of k13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative RDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin partial resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.

Ten-year trend analysis of malaria prevalence in Gindabarat district, West Shawa Zone, Oromia Regional State, Western Ethiopia.

BACKGROUND: Malaria is a major public health concern in Ethiopia, where more than half of the population lives in malaria risk areas. While several studies have been conducted in different eco-epidemiological settings in Ethiopia, there is a notable scarcity of data on the prevalence of malaria in the Gindabarat district. Therefore, this study aimed to analyse 10-year trend of malaria prevalence in Gindabarat district, West Shawa Zone of Oromia, Western Ethiopia. METHODS: A retrospective laboratory record review was conducted at Gindabarat General Hospital and Gindabarat District Health Office from September 2011 to August 2020. The retrieved data included the date of examination, age, sex and laboratory results of the blood smears, including the Plasmodium species identified. Data were summarized and presented in the form of tables, figures, and frequencies to present the results. The data were analysed using SPSS (version 25.0) and Microsoft Excel. RESULTS: Over the course of 10 years, a total of 11,478 blood smears were examined in the public health facilities in the district. Of the total blood smears examined, 1372 (11.95%) were microscopically confirmed malaria. Plasmodium falciparum, Plasmodium vivax and mixed infections (P. falciparum and P. vivax) accounted for 70.77%, 20.55% and 8.67% of the cases, respectively. Malaria prevalence was significantly higher among individuals aged ≥ 15 years (12.60%, x2 = 13.6, df = 2, p = 0.001) and males (14.21%, x2 = 59.7, df = 1, p = 0.001). The highest number of malaria cases was recorded from September to November. CONCLUSION: Malaria remains a public health problem in the district. P. falciparum was the most predominant parasite species in the area. Malaria prevalence was significantly higher among individuals aged ≥ 15 years and males. There was a remarkable fluctuation in the number of malaria cases in different months and years. In the study area malaria cases peaked in 2015 and 2017 then decreasing from 2017 to 2019, with sharp increase in 2020. Moreover, this study showed malaria cases were reported in all seasons and months, but the highest was observed from September to November. Strengthening malaria control activities is essential to further reduce the burden of malaria and pave the way for the anticipated elimination.

Severe outcomes of malaria in children under time-varying exposure.

In malaria epidemiology, interpolation frameworks based on available observations are critical for policy decisions and interpreting disease burden. Updating our understanding of the empirical evidence across different populations, settings, and timeframes is crucial to improving inference for supporting public health. Here, via individual-based modeling, we evaluate a large, multicountry, contemporary Plasmodium falciparum severe malaria dataset to better understand the relationship between prevalence and incidence of malaria pediatric hospitalizations - a proxy of malaria severe outcomes- in East-Africa. We find that life-long exposure dynamics, and subsequent protection patterns in children, substantially determine the likelihood of malaria hospitalizations relative to ongoing prevalence at the population level. Unsteady transmission patterns over a lifetime in children -increasing or decreasing- lead to an exponential relationship of hospitalization rates versus prevalence rather than the asymptotic pattern observed under steady transmission. Addressing this increase in the complexity of malaria epidemiology is crucial to update burden assessments via inference models that guide current and future policy decisions.

Comparison of malaria diagnostic methods for detection of asymptomatic Plasmodium infections among pregnant women in northwest Ethiopia.

BACKGROUND: Malaria in pregnancy remains a major public health problem in the globe, especially in sub-Saharan Africa. In malaria endemic areas, most pregnant women remain asymptomatic, but malaria could still cause complications on the mother and her offspring; as well as serve as reservoirs to transmit infection. Despite these effects, no attention is given to the diagnosis of asymptomatic Plasmodium infections (APIs) using highly sensitive and specific laboratory diagnostic tools in Ethiopia. Therefore, the goal of this study was to compare the performance of Rapid Diagnostic Test (RDT), microscopy and real-time polymerase chain reaction (RT-PCR) to detect APIs among pregnant women. METHODS: A health facility based cross -sectional study was conducted among pregnant women attending antenatal care at Fendeka town health facilities Jawi district, northwest Ethiopia from February to March, 2019. A total of 166 participants were enrolled by using convenient sampling technique. Socio-demographic features were collected using a semi structured questionnaire. Dried blood spot (DBS) samples were collected for molecular analysis. Asymptomatic Plasmodium infection on pregnant women was diagnosed using RDT, microscopy and RT-PCR. Descriptive statistics were used to determine the prevalence of APIs. Method comparison was performed, and Cohen's kappa coefficient (k) was used to determine the degree of agreement among the diagnostic methods. Parasite densities were also calculated. RESULTS: The prevalence of API was 9.6%, 11.4% and 18.7% using RDT, microscopy and RT-PCR, respectively. The overall proportion of API was 19.3%. Sensitivity of the RDT was 83.3% as compared with microscopy. Rapid Diagnostic Test and microscopy also showed sensitivity of 50% and 60%, respectively, as compared with RT-PCR. The mean parasite density was 3213 parasites/µl for P falciparum and 1140 parasites/µl of blood for P. vivax. CONCLUSION: Prevalence of API in the study area was high. Both RDT and microscopy had lower sensitivity when compared with RT-PCR. Therefore, routine laboratory diagnosis of API among pregnant women should be given attention and done with better sensitive and specific laboratory diagnostic tools.

Genetic polymorphism and evidence of signatures of selection in the Plasmodium falciparum circumsporozoite protein gene in Tanzanian regions with different malaria endemicity.

BACKGROUND: In 2021 and 2023, the World Health Organization approved RTS,S/AS01 and R21/Matrix M malaria vaccines, respectively, for routine immunization of children in African countries with moderate to high transmission. These vaccines are made of Plasmodium falciparum circumsporozoite protein (PfCSP), but polymorphisms in the gene raise concerns regarding strain-specific responses and the long-term efficacy of these vaccines. This study assessed the Pfcsp genetic diversity, population structure and signatures of selection among parasites from areas of different malaria transmission intensities in Mainland Tanzania, to generate baseline data before the introduction of the malaria vaccines in the country. METHODS: The analysis involved 589 whole genome sequences generated by and as part of the MalariaGEN Community Project. The samples were collected between 2013 and January 2015 from five regions of Mainland Tanzania: Morogoro and Tanga (Muheza) (moderate transmission areas), and Kagera (Muleba), Lindi (Nachingwea), and Kigoma (Ujiji) (high transmission areas). Wright's inbreeding coefficient (Fws), Wright's fixation index (FST), principal component analysis, nucleotide diversity, and Tajima's D were used to assess within-host parasite diversity, population structure and natural selection. RESULTS: Based on Fws (< 0.95), there was high polyclonality (ranging from 69.23% in Nachingwea to 56.9% in Muheza). No population structure was detected in the Pfcsp gene in the five regions (mean FST = 0.0068). The average nucleotide diversity (π), nucleotide differentiation (K) and haplotype diversity (Hd) in the five regions were 4.19, 0.973 and 0.0035, respectively. The C-terminal region of Pfcsp showed high nucleotide diversity at Th2R and Th3R regions. Positive values for the Tajima's D were observed in the Th2R and Th3R regions consistent with balancing selection. The Pfcsp C-terminal sequences revealed 50 different haplotypes (H_1 to H_50), with only 2% of sequences matching the 3D7 strain haplotype (H_50). Conversely, with the NF54 strain, the Pfcsp C-terminal sequences revealed 49 different haplotypes (H_1 to H_49), with only 0.4% of the sequences matching the NF54 strain (Hap_49). CONCLUSIONS: The findings demonstrate high diversity of the Pfcsp gene with limited population differentiation. The Pfcsp gene showed positive Tajima's D values, consistent with balancing selection for variants within Th2R and Th3R regions. The study observed differences between the intended haplotypes incorporated into the design of RTS,S and R21 vaccines and those present in natural parasite populations. Therefore, additional research is warranted, incorporating other regions and more recent data to comprehensively assess trends in genetic diversity within this important gene. Such insights will inform the choice of alleles to be included in the future vaccines.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.