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.

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.

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.

Identification of domains in Plasmodium falciparum proteins of unknown function using DALI search on AlphaFold predictions.

Plasmodium falciparum, the causative agent of malaria, poses a significant global health challenge, yet much of its biology remains elusive. A third of the genes in the P. falciparum genome lack annotations regarding their function, impeding our understanding of the parasite's biology. In this study, we employ structure predictions and the DALI search algorithm to analyse proteins encoded by uncharacterized genes in the reference strain 3D7 of P. falciparum. By comparing AlphaFold predictions to experimentally determined protein structures in the Protein Data Bank, we found similarities to known domains in 353 proteins of unknown function, shedding light on their potential functions. The lowest-scoring 5% of similarities were additionally validated using the size-independent TM-align algorithm, confirming the detected similarities in 88% of the cases. Notably, in over 70 P. falciparum proteins the presence of domains resembling heptatricopeptide repeats, which are typically involvement in RNA binding and processing, was detected. This suggests this family, which is important in transcription in mitochondria and apicoplasts, is much larger in Plasmodium parasites than previously thought. The results of this domain search provide a resource to the malaria research community that is expected to inform and enable experimental studies.

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.

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.

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.

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.

The prevalence of Pfk13 polymorphism in malaria patients treated with artemisinin-based therapy: a systematic review and meta-analysis.

Artemisinin (ART) combination therapy is the main treatment for malaria. Pfk13 mutations (or K13 mutations, Kelch 13) are associated with ART resistance. This study aims to conduct a systematic review and meta-analysis of the prevalence of K13 mutations with ART resistance in malaria-endemic countries. An electronic search of studies in 2018 and a manual search in 2020 were performed to identify relevant studies. The risk of bias was assessed using the National Institutes of Health (NIH) quality assessment tool for observational cohort and cross-sectional studies. Data analysis was performed using R 4.1.0. Heterogeneity was estimated using the statistic I2 and Cochran Q test. A total of 170 studies were included in our review. Of these, 55 studies investigated the prevalence of K13 mutations in Southeast Asia. The meta-analysis showed that Southeast Asia had the highest prevalence of K13 mutations, whereas Africa, South America, Oceania, and other Asian countries outside Southeast Asia had a low prevalence of K13 mutations. The C580Y mutation was the most common in Southeast Asia with 35.5% (95%CI: 25.4-46.4%), whereas the dominant mutation in Africa was K189T (22.8%, 95%CI: 7.6-43.2%). This study revealed the emergence of ART resistance associated with K13 mutations in Southeast Asia. The diversity of each type of K13 mutation in other regions was also reported.

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.

Therapeutic efficacy and tolerability of artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria in Niger, 2020.

BACKGROUND: Monitoring therapeutic efficacy is important to ensure the efficacy of artemisinin-based combination therapy (ACT) for malaria. The current first-line treatment for uncomplicated malaria recommended by the National Malaria Control Program in Niger is artemether-lumefantrine (AL). In 2020, an in vivo study was carried out to evaluate clinical and parasitological responses to AL as well as the molecular resistance to the drug in three sentinel sites: Agadez, Tessaoua and Gaya, in Niger. METHODS: A multi-center, single-arm trial was conducted according to the 28-day World Health Organization (WHO) 2009 therapeutic efficacy study protocol. Children between 6 months and 15 years with confirmed uncomplicated Plasmodium falciparum infection and 1000-200,000 asexual parasites/µL of blood were enrolled and followed up for 28 days. Uncorrected and PCR-corrected efficacy results at day 28 were calculated, and molecular correction was performed by genotyping the msp1, msp2, and glurp genes. The pfk13, pfdhfr, pfdhps, pfcrt and pfmdr genes were analyzed by PCR and Sanger sequencing. The Kaplan-Meier curve assessed parasite clearance. RESULTS: A total of 255 patients were enrolled in the study. The adequate clinical and parasitological response after PCR correction was 98.9% (95% CI 96.4-101.0%), 92.2% (85.0-98.5%) and 97.1% (93.1-101.0%) in Gaya, Tessaoua and Agadez, respectively. No adverse events were observed. Ten mutations (SNP) were found, including 7 synonyms (K248K, G690G, E691E, E612E, C469C, G496G, P718P) and 3 non-synonyms (N594K, R255K, V714S). Two mutations emerged: N594K and V714S. The R255K mutation detected in Southeast Asia was also detected. The pfdhpsK540E and pfdhfrI164L mutations associated with high levels of resistance are absent. There is a reversal of chloroquine resistance. CONCLUSION: The study findings indicate that AL is effective and well tolerated for the treatment of uncomplicated malaria in three sites in Niger. The emergence of a pfk13 mutation requires additional testing such as the Ring Stage Assay and CRISPR/Cas9 to confirm the role of these emerging mutations. Trial registration NCT05070520, October 7, 2021.

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.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host-pathogen interactions.

BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

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%).

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.

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.

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.

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites. IMPORTANCE: Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

Positive-unlabeled learning identifies vaccine candidate antigens in the malaria parasite Plasmodium falciparum.

Malaria vaccine development is hampered by extensive antigenic variation and complex life stages of Plasmodium species. Vaccine development has focused on a small number of antigens, many of which were identified without utilizing systematic genome-level approaches. In this study, we implement a machine learning-based reverse vaccinology approach to predict potential new malaria vaccine candidate antigens. We assemble and analyze P. falciparum proteomic, structural, functional, immunological, genomic, and transcriptomic data, and use positive-unlabeled learning to predict potential antigens based on the properties of known antigens and remaining proteins. We prioritize candidate antigens based on model performance on reference antigens with different genetic diversity and quantify the protein properties that contribute most to identifying top candidates. Candidate antigens are characterized by gene essentiality, gene ontology, and gene expression in different life stages to inform future vaccine development. This approach provides a framework for identifying and prioritizing candidate vaccine antigens for a broad range of pathogens.

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.