The Plasmodium falciparum CCCH zinc finger protein MD3 regulates male gametocytogenesis through its interaction with RNA-binding proteins.

The transmission of malaria parasites to mosquitoes is dependent on the formation of gametocytes. Once fully matured, gametocytes are able to transform into gametes in the mosquito's midgut, a process accompanied with their egress from the enveloping erythrocyte. Gametocyte maturation and gametogenesis require a well-coordinated gene expression program that involves a wide spectrum of regulatory proteins, ranging from histone modifiers to transcription factors to RNA-binding proteins. Here, we investigated the role of the CCCH zinc finger protein MD3 in Plasmodium falciparum gametocytogenesis. MD3 was originally identified as an epigenetically regulated protein of immature gametocytes and recently shown to be involved in male development in a barcode-based screen in P. berghei. We report that MD3 is mainly present in the cytoplasm of immature male P. falciparum gametocytes. Parasites deficient of MD3 are impaired in gametocyte maturation and male gametocytogenesis. BioID analysis in combination with co-immunoprecipitation assays unveiled an interaction network of MD3 with RNA-binding proteins like PABP1 and ALBA3, with translational initiators, regulators and repressors like elF4G, PUF1, NOT1 and CITH, and with further regulators of gametocytogenesis, including ZNF4, MD1 and GD1. We conclude that MD3 is part of a regulator complex crucial for post-transcriptional fine-tuning of male gametocytogenesis.

Ongoing mpox outbreak in Kamituga, South Kivu province, associated with monkeypox virus of a novel Clade I sub-lineage, Democratic Republic of the Congo, 2024.

Since the beginning of 2023, the number of people with suspected monkeypox virus (MPXV) infection have sharply increased in the Democratic Republic of the Congo (DRC). We report near-to-complete MPXV genome sequences derived from six cases from the South Kivu province. Phylogenetic analyses reveal that the MPXV affecting the cases belongs to a novel Clade I sub-lineage. The outbreak strain genome lacks the target sequence of the probe and primers of a commonly used Clade I-specific real-time PCR.

False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission.

BACKGROUND: Rapid diagnostic tests (RDTs) for histidine rich protein 2 (HRP2) are often used to determine whether persons with fever should be treated with anti-malarials. However, Plasmodium falciparum parasites with a deletion of the hrp2 gene yield false-negative RDTs and there are concerns the sensitivity of HRP2-based RDTs may fall when the intensity of transmission decreases. METHODS: This observational study enrolled 9226 patients at three health centres in Rwanda from April 2014 to April 2015. It then compared the sensitivity of RDTs based on HRP2 and the Plasmodium lactate dehydrogenase (pLDH) to microscopy (thick smears) for the diagnosis of malaria. PCR was used to determine whether deletions of the histidine-rich central repeat region of the hrp2 gene (exon 2) were associated with false-negative HRP2-based RDTs. RESULTS: In comparison to microscopy, the sensitivity and specificity of HRP2- and pLDH-based RDTs were 89.5 and 86.2% and 80.2 and 94.3%, respectively. When the results for both RDTs were combined, sensitivity rose to 91.8% and specificity was 85.7%. Additionally, when smear positivity fell from 46 to 3%, the sensitivity of the HRP2-based RDT fell from 88 to 67%. Of 370 samples with false-negative HRP2 RDT results for which PCR was performed, 140 (38%) were identified as P. falciparum by PCR. Of the isolates identified as P. falciparum by PCR, 32 (23%) were negative for the hrp2 gene based on PCR. Of the 32 P. falciparum isolates negative for hrp2 by PCR, 17 (53%) were positive based on the pLDH RDT. CONCLUSION: This prospective study of RDT performance coincided with a decline in the intensity of malaria transmission in Kibirizi (fall in slide positivity from 46 to 3%). This decline was associated with a decrease in HRP2 RDT sensitivity (from 88 to 67%). While P. falciparum isolates without the hrp2 gene were an important cause of false-negative HRP2-based RDTs, most were identified by the pLDH-based RDT. Although WHO does not recommend the use of combined HRP2/pLDH testing in sub-Saharan Africa, these results suggest that combination HRP2/pLDH-based RDTs could reduce the impact of false-negative HRP2-based RDTs for detection of symptomatic P. falciparum malaria.

The Plasmodium falciparum histone methyltransferase SET10 participates in a chromatin modulation network crucial for intraerythrocytic development.

The lifecycle progression of the malaria parasite Plasmodium falciparum requires precise tuning of gene expression including histone methylation. The histone methyltransferase PfSET10 was previously described as an H3K4 methyltransferase involved in var gene regulation, making it a prominent antimalarial target. In this study, we investigated the role of PfSET10 in the blood stages of P. falciparum in more detail, using tagged PfSET10-knockout (KO) and -knockdown (KD) lines. We demonstrate a nuclear localization of PfSET10 with peak protein levels in schizonts. PfSET10 deficiency reduces intraerythrocytic growth but has no effect on gametocyte commitment and maturation. Screening of the PfSET10-KO line for histone methylation variations reveals that lack of PfSET10 renders the parasites unable to mark H3K18me1, while no reduction in the H3K4 methylation status could be observed. Comparative transcriptomic profiling of PfSET10-KO schizonts shows an upregulation of transcripts particularly encoding proteins linked to red blood cell remodeling and antigenic variation, suggesting a repressive function of the histone methylation mark. TurboID coupled with mass spectrometry further highlights an extensive nuclear PfSET10 interaction network with roles in transcriptional regulation and mRNA processing, DNA replication and repair, and chromatin remodeling. The main interactors of PfSET10 include ApiAP2 transcription factors, epigenetic regulators like PfHDAC1, chromatin modulators like PfMORC and PfISWI, mediators of RNA polymerase II, and DNA replication licensing factors. The combined data pinpoint PfSET10 as a histone methyltransferase essential for H3K18 methylation that regulates nucleic acid metabolic processes in the P. falciparum blood stages as part of a comprehensive chromatin modulation network.IMPORTANCEThe fine-tuned regulation of DNA replication and transcription is particularly crucial for the rapidly multiplying blood stages of malaria parasites and proteins involved in these processes represent important drug targets. This study demonstrates that contrary to previous reports the histone methyltransferase PfSET10 of the malaria parasite Plasmodium falciparum promotes the methylation of histone 3 at lysine K18, a histone mark to date not well understood. Deficiency of PfSET10 due to genetic knockout affects genes involved in intraerythrocytic development. Furthermore, in the nuclei of blood-stage parasites, PfSET10 interacts with various protein complexes crucial for DNA replication, remodeling, and repair, as well as for transcriptional regulation and mRNA processing. In summary, this study highlights PfSET10 as a methyltransferase affecting H3K18 methylation with critical functions in chromatin maintenance during the development of P. falciparum in red blood cells.

Effect of unmanned aerial vehicle (drone) delivery on blood product delivery time and wastage in Rwanda: a retrospective, cross-sectional study and time series analysis.

BACKGROUND: The accessibility of blood and blood products remains challenging in many countries because of the complex supply chain of short lifetime products, timely access, and demand fluctuation at the hospital level. In an effort to improve availability and delivery times, Rwanda launched the use of drones to deliver blood products to remote health facilities. We evaluated the effect of this intervention on blood product delivery times and wastage. METHODS: We studied data from 20 health facilities between Jan 1, 2015, and Dec 31, 2019, in Rwanda. First, we did a cross-sectional comparison of data on emergency delivery times from the drone operator collected between March 17, 2017, and Dec 31, 2019, with two sources of estimated driving times (Regional Centre for Blood Transfusion estimates and Google Maps). Second, we used interrupted time series analysis and monthly administrative data to assess changes in blood product expirations after the commencement of drone deliveries. FINDINGS: Between March 17, 2017, and Dec 31, 2019, 12 733 blood product orders were delivered by drones. 5517 (43%) of 12 733 were emergency orders. The mean drone delivery time was 49·6 min (95% CI 49·1 to 50·2), which was 79 min faster than existing road delivery methods based on estimated driving times (p<0·0001) and 98 min faster based on Google Maps estimates (p<0·0001). The decrease in mean delivery time ranged from 3 min to 211 min depending on the distance to the facility and road quality. We also found a decrease of 7·1 blood unit expirations per month after the start of drone delivery (95% CI -11·8 to -2·4), which translated to a 67% reduction at 12 months. INTERPRETATION: We found that drone delivery led to faster delivery times and less blood component wastage in health facilities. Future studies should investigate if these improvements are cost-effective, and whether drone delivery might be effective for other pharmaceutical and health supplies that cannot be easily stored at remote facilities. FUNDING: Canadian Institutes for Health Research.

Evaluation of Chiral Organosulfur Compounds on Their Activity against the Malaria Parasite Plasmodium falciparum.

Malaria is one of the deadliest tropical diseases, especially causing havoc in children under the age of five in Africa. Although the disease is treatable, the rapid development of drug resistant parasites against frontline drugs requires the search for novel antimalarials. In this study, we tested a series of organosulfur compounds from our internal library for their antiplasmodial effect against Plasmodium falciparum asexual and sexual blood stages. Some active compounds were also obtained in enantiomerically pure form and tested individually against asexual blood stages of the parasite to compare their activity. Out of the 23 tested compounds, 7 compounds (1, 2, 5, 9, 15, 16, and 17) exhibited high antimalarial activity, with IC50 values in the range from 2.2 ± 0.64 to 5.2 ± 1.95 µM, while the other compounds showed moderate to very low activity. The most active compounds also exhibited high activity against the chloroquine-resistant strain, reduced gametocyte development and were not toxic to non-infected red blood cells and Hela cells, as well as the hematopoietic HEL cell line at concentrations below 50 µM. To determine if the enantiomers of the active compounds display different antimalarial activity, enantiomers of two of the active compounds were separated and their antimalarial activity compared. The results show a higher activity of the (-) enantiomers as compared to their (+) counterparts. Our combined data indicate that organosulfur compounds could be exploited as antimalarial drugs and enantiomers of the active compounds may represent a good starting point for the design of novel drugs to target malaria.

Plasmodium falciparum S-Adenosylmethionine Synthetase Is Essential for Parasite Survival through a Complex Interaction Network with Cytoplasmic and Nuclear Proteins.

S-adenosylmethionine synthetase (SAMS) is a key enzyme for the synthesis of the lone methyl donor S-adenosyl methionine (SAM), which is involved in transmethylation reactions and hence required for cellular processes such as DNA, RNA, and histone methylation, but also polyamine biosynthesis and proteostasis. In the human malaria parasite Plasmodium falciparum, PfSAMS is encoded by a single gene and has been suggested to be crucial for malaria pathogenesis and transmission; however, to date, PfSAMS has not been fully characterized. To gain deeper insight into the function of PfSAMS, we generated a conditional gene knockdown (KD) using the glmS ribozyme system. We show that PfSAMS localizes to the cytoplasm and the nucleus of blood-stage parasites. PfSAMS-KD results in reduced histone methylation and leads to impaired intraerythrocytic growth and gametocyte development. To further determine the interaction network of PfSAMS, we performed a proximity-dependent biotin identification analysis. We identified a complex network of 1114 proteins involved in biological processes such as cell cycle control and DNA replication, or transcription, but also in phosphatidylcholine and polyamine biosynthesis and proteasome regulation. Our findings highlight the diverse roles of PfSAMS during intraerythrocytic growth and sexual stage development and emphasize that PfSAMS is a potential drug target.

The Role of the Histone Methyltransferase PfSET10 in Antigenic Variation by Malaria Parasites: a Cautionary Tale.

The virulence of the malaria parasite Plasmodium falciparum is due in large part to its ability to avoid immune destruction through antigenic variation. This results from changes in expression within the multicopy var gene family that encodes the surface antigen P. falciparum erythrocyte protein one (PfEMP1). Understanding the mechanisms underlying this process has been a high-profile research focus for many years. The histone methyltransferase PfSET10 was previously identified as a key enzyme required both for parasite viability and for regulating var gene expression, thus making it a prominent target for developing antimalarial intervention strategies and the subject of considerable research focus. Here, however, we show that disruption of the gene encoding PfSET10 is not lethal and has no effect on var gene expression, in sharp contrast with previously published reports. The contradictory findings highlight the importance of reevaluating previous conclusions when new technologies become available and suggest the possibility of a previously unappreciated plasticity in epigenetic gene regulation in P. falciparumIMPORTANCE The identification of specific epigenetic regulatory proteins in infectious organisms has become a high-profile research topic and a focus for several drug development initiatives. However, studies that define specific roles for different epigenetic modifiers occasionally report differing results, and we similarly provide evidence regarding the histone methyltransferase PfSET10 that is in stark contrast with previously published results. We believe that the conflicting results, rather than suggesting erroneous conclusions, instead reflect the importance of revisiting previous conclusions using newly developed methodologies, as well as caution in interpreting seemingly contrary results in fields that are known to display considerable plasticity, for example metabolism and epigenetics.

Prevalence of human respiratory syncytial virus, parainfluenza and adenoviruses in East Africa Community partner states of Kenya, Tanzania, and Uganda: A systematic review and meta-analysis (2007-2020).

BACKGROUND: Viruses are responsible for a large proportion of acute respiratory tract infections (ARTIs). Human influenza, parainfluenza, respiratory-syncytial-virus, and adenoviruses are among the leading cause of ARTIs. Epidemiological evidence of those respiratory viruses is limited in the East Africa Community (EAC) region. This review sought to identify the prevalence of respiratory syncytial virus, parainfluenza, and adenoviruses among cases of ARTI in the EAC from 2007 to 2020. METHODS: A literature search was conducted in Medline, Global Index Medicus, and the grey literature from public health institutions and programs in the EAC. Two independent reviewers performed data extraction. We used a random effects model to pool the prevalence estimate across studies. We assessed heterogeneity with the I2 statistic, and Cochran's Q test, and further we did subgroup analysis. This review was registered with PROSPERO under registration number CRD42018110186. RESULTS: A total of 12 studies met the eligibility criteria for the studies documented from 2007 to 2020. The overall pooled prevalence of adenoviruses was 13% (95% confidence interval [CI]: 6-21, N = 28829), respiratory syncytial virus 11% (95% CI: 7-15, N = 22627), and parainfluenza was 9% (95% CI: 7-11, N = 28363). Pooled prevalence of reported ARTIs, all ages, and locality varied in the included studies. Studies among participants with severe acute respiratory disease had a higher pooled prevalence of all the three viruses. Considerable heterogeneity was noted overall and in subgroup analysis. CONCLUSION: Our findings indicate that human adenoviruses, respiratory syncytial virus and parainfluenza virus are prevalent in Kenya, Tanzania, and Uganda. These three respiratory viruses contribute substantially to ARTIs in the EAC, particularly among those with severe disease and those aged five and above.

Few Plasmodium falciparum merozoite ligand and erythrocyte receptor pairs show evidence of balancing selection.

Erythrocyte surface proteins have been identified as receptors of Plasmodium falciparum merozoite proteins. The ligand-receptor interactions enable the parasite to invade human erythrocytes, initiating the clinical symptoms of malaria. These interactions are likely to have had an evolutionary impact on the genes that encode the ligand and receptor proteins. We used sequence data from Kilifi, Kenya to detect departures from neutrality in a paired analysis of P. falciparum merozoite ligands and their erythrocyte receptor genes from the same population. We genotyped parasite and human DNA obtained from 93 individuals with severe malaria. We examined six merozoite ligands EBA175, EBL1, EBA140, MSP1, Rh4 and Rh5, and their corresponding erythrocyte receptors, glycophorin (Gyp) A, GypB, GypC, band 3, complement receptor (CR) 1 and basigin, focusing on the regions involved in the ligand-receptor interactions. Positive Tajima's D values (>1) were observed only in the MSP1 C-terminal region and EBA175 region II, while negative values (<-1) were observed in EBL-1 region II, Rh4, basigin exons 3 and 5, CR1 exon 5, Gyp B exons 2, 3 and 4 and Gyp C exon 2. Additionally, ebl-1 region II and basigin exon 3 showed extreme negative values in all three tests, Tajima's D, Fu & Li D* and F*, ≤ - 2. A large majority of the erythrocyte receptor and merozoite genes have a negative Tajima's D even when compared with previously published whole genome data. Thus, highlighting EBA175 region II and MSP1-33, as outlier genes with a positive Tajima's D (>1). Both these genes contain multiple polymorphisms, which in the case of EBA175 may counteract receptor polymorphisms and/or evade host immune responses and in MSP1 the polymorphisms may primarily evade host immune responses.

A One Health systematic review of diagnostic tools for Echinococcus multilocularis surveillance: Towards equity in global detection.

Echinococcus multilocularis is a zoonotic cestode of canid definitive hosts that is emerging as a parasite of medical and veterinary concern in regions of North America, Europe and Asia. Infection with the metacestode stage (alveolar echinococcosis - AE) is life-threatening, especially for patients who reside in low resource countries and lack access to modern diagnostic tests and treatments. The overall objectives of this One Health review were to systematically describe the diagnostic tests currently employed in endemic countries to detect E. multilocularis in people, canids and the environment, and to report the test characteristics of new diagnostic techniques for population surveillance. In this systematic review of English and Chinese language databases, we identified 92 primary records of E. multilocularis surveillance in canids (N = 75), humans (N = 20) and/or the environment (food, soil; N = 3) and 12 grey literature records that reported E. multilocularis surveillance or health systems protocols between 2008 and 2018. Surveillance for E. multilocularis was conducted using a broad range of combined morphological, molecular, immunological and imaging techniques. Nine studies reporting diagnostic evaluations for cestode or metacestode detection were identified, including studies on copro-antigen ELISA, copro-PCR, intestinal examination, Western Blot, magnetic capture RT-PCR and immunochromatography. Our dataset includes prevalence estimates for E. multilocularis in canids, people, or environment in 27 of the 43 endemic countries and reports data gaps in surveillance, laboratory methods, and diagnostic sensitivity. International consensus on gold standard diagnostic techniques and harmonization of human, canid and environmental surveillance data across political boundaries are needed to comprehensively assess the global burden and distribution of this parasite.

Expanding home-based management of malaria to all age groups in Rwanda: analysis of acceptability and facility-level time-series data.

Background: In response to a resurgence of malaria in Rwanda, home-based management (HBM) was expanded to enable community-health workers (CHWs) to provide malaria treatment to patients of all ages. We assessed the effect of the expanded HBM program on malaria case presentations at health facilities. Methods: Services provided by CHWs and health facility presentations among individuals >5 y of age were considered. Presentations to CHWs were analyzed descriptively to assess acceptability and segmented regression modeling using facility-level data was employed to compare changes between the pre- and postintervention periods for intervention and control districts. Results: Individuals >5 y of age readily accessed malaria diagnosis and treatment services from CHWs. Severe and uncomplicated malaria increased in the postintervention period for both the intervention and control districts. Presentations for uncomplicated malaria increased in the intervention and control districts to a similar degree. Severe cases increased to a greater degree in the intervention districts immediately after HBM was expanded compared with controls, but the monthly rate of increase was lower in the intervention districts. Conclusions: Services were shifted to CHWs, as demonstrated by the number of individuals treated through the expanded program. The rate of severe malaria increased immediately after implementation within intervention districts relative to controls, potentially because of enhanced case-finding. The rate of increase in severe cases was lower in the intervention districts comparatively, likely due to expedited treatment.