An axonemal intron splicing program sustains Plasmodium male development.

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites.

BACKGROUND: Natural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. METHODS: By using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. RESULTS: There was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. CONCLUSION: This is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

Zombie malaria parasites.

Natural infections are distinct from those of laboratory-or zombie-strains.

Dual CRISPR/Cas13a Cascade Strand Displacement-Triggered Transcription for Point-of-Care Detection of Plasmodium in Asymptomatic Malaria.

Asymptomatic infections of Plasmodium parasites are major obstacles to malaria control and elimination. A sensitive, specific, and user-friendly method is urgently needed for point-of-care (POC) Plasmodium diagnostics in asymptomatic malaria, especially in resource-limited settings. In this work, we present a POC method (termed Cas13a-SDT) based on the cascade sequence recognition and signal amplification of dual Cas13a trans-cleavage and strand displacement-triggered transcription (SDT). Cas13a-SDT not only achieves exceptional specificity in discriminating the target RNA from nontarget RNAs with any cross-interaction but also meets the sensitivity criterion set by the World Health Organization (WHO) for effective malaria detection. Remarkably, this novel method was successfully applied to screen malaria in asymptomatic infections from clinical samples. The proposed method provides a user-friendly and visually interpretable output mode while maintaining high accuracy and reliability comparable to RT-PCR. These excellent features demonstrate the significant potential of Cas13a-SDT for POC diagnosis of Plasmodium infections, laying a vital foundation for advancing malaria control and elimination efforts.

Molecular prevalence, phylogeny and hematological impact of Toxoplasma gondii and Plasmodium spp. in common quails from Punjab, Pakistan.

This study investigates the molecular prevalence and phylogenetic characteristics of two prominent blood-borne pathogens, Toxoplasma gondii (T. gondii) and Plasmodium spp., in common quails (Coturnix coturnix) sampled from both wild (N = 236) and farmed (N = 197) populations across four districts (Layyah, Dera Ghazi Khan, Lahore, and Multan) in Punjab, Pakistan, during the hunting seasons from 2021 to 2023. Additionally, the impact of these pathogens on the complete blood count (CBC) of the hosts is examined. Out of 433 quails tested, 25 (5.8%) exhibited amplification of the internal transcribed spacer (ITS-1) gene for T. gondii, while 15 (3.5%) showed amplification of the Cytochrome b gene for Plasmodium spp. A risk factor analysis indicated that the prevalence of both pathogens was not confined to specific sampling sites or bird sexes (P > 0.05). District-wise analysis highlighted that hens were more susceptible to both T. gondii and Plasmodium spp. infections than cocks. Wild quails exhibited a higher susceptibility to T. gondii compared to farmed birds. Significant CBC variations were recorded in infected birds as compared to uninfected ones. BLAST analysis of generated sequences has confirmed the identity of recovered PCR amplicons as T. gondii and Plasmodium relictum. Phylogenetic analysis revealed that Pakistani isolates clustered with those reported from various countries globally. This study provides the first documentation of T. gondii and Plasmodium sp. infections in Pakistani quails, underscoring the need for detailed investigations across different regions to enhance our understanding of infection rates and the zoonotic potential of these parasites.

Exploring mosquito abundance and Plasmodium infection through nested-PCR: implications for disease surveillance and control.

The Plasmodium is responsible for malaria which poses a major health threat, globally. This study is based on the estimation of the relative abundance of mosquitoes, and finding out the correlations of meteorological parameters (temperature, humidity and rainfall) with the abundance of mosquitoes. In addition, this study also focused on the use of nested PCR (species-specific nucleotide sequences of 18S rRNA genes) to explore the Plasmodium spp. in female Anopheles. In the current study, the percentage relative abundance of Culex mosquitoes was 57.65% and Anopheles 42.34% among the study areas. In addition, the highest number of mosquitoes was found in March in district Mandi Bahauddin at 21 °C (Tmax = 27, Tmin = 15) average temperature, 69% average relative humidity and 131 mm rainfall, and these climatic factors were found to affect the abundance of the mosquitoes, directly or indirectly. Molecular analysis showed that overall, 41.3% of the female Anopheles pools were positive for genus Plasmodium. Among species, the prevalence of Plasmodium (P.) vivax (78.1%) was significantly higher than P. falciparum (21.9%). This study will be helpful in the estimation of future risk of mosquito-borne diseases along with population dynamic of mosquitoes to enhance the effectiveness of vector surveillance and control programs.

Genotype-environment associations reveal genes potentially linked to avian malaria infection in populations of an endemic island bird.

Patterns of pathogen prevalence are, at least partially, the result of coevolutionary host-pathogen interactions. Thus, exploring the distribution of host genetic variation in relation to infection by a pathogen within and across populations can provide important insights into mechanisms of host defence and adaptation. Here, we use a landscape genomics approach (Bayenv) in conjunction with genome-wide data (ddRADseq) to test for associations between avian malaria (Plasmodium) prevalence and host genetic variation across 13 populations of the island endemic Berthelot's pipit (Anthus berthelotii). Considerable and consistent spatial heterogeneity in malaria prevalence was observed among populations over a period of 15 years. The prevalence of malaria infection was also strongly positively correlated with pox (Avipoxvirus) prevalence. Multiple host loci showed significant associations with malaria prevalence after controlling for genome-wide neutral genetic structure. These sites were located near to or within genes linked to metabolism, stress response, transcriptional regulation, complement activity and the inflammatory response, many previously implicated in vertebrate responses to malarial infection. Our findings identify diverse genes - not just limited to the immune system - that may be involved in host protection against malaria and suggest that spatially variable pathogen pressure may be an important evolutionary driver of genetic divergence among wild animal populations, such as Berthelot's pipit. Furthermore, our data indicate that spatio-temporal variation in multiple different pathogens (e.g. malaria and pox in this case) may have to be studied together to develop a more holistic understanding of host pathogen-mediated evolution.

New insight into avian malaria vectors in New Zealand.

BACKGROUND: Mosquitoes (Culicidae) are vectors for most malaria parasites of the Plasmodium species and are required for Plasmodium spp. to complete their life cycle. Despite having 16 species of mosquitoes and the detection of many Plasmodium species in birds, little is known about the role of different mosquito species in the avian malaria life cycle in New Zealand. METHODS: In this study, we used nested polymerase chain reaction (PCR) and real-time PCR to determine Plasmodium spp. prevalence and diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across ten sites on the North Island of New Zealand during 2012-2014. The mosquitoes were pooled by species and location collected, and the thorax and abdomens were examined separately for Plasmodium spp. DNA. Akaike information criterion (AIC) modeling was used to test whether location, year of sampling, and mosquito species were significant predictors of minimum infection rates (MIR). RESULTS: We collected 788 unengorged mosquitoes of six species, both native and introduced. The most frequently caught mosquito species were the introduced Aedes notoscriptus and the native Culex pervigilans. Plasmodium sp DNA was detected in 37% of matched thorax and abdomen pools. When considered separately, 33% of abdomen and 23% of thorax pools tested positive by nested PCR. The MIR of the positive thorax pools from introduced mosquito species was 1.79% for Ae. notoscriptus and 0% for Cx. quinquefasciatus, while the MIR for the positive thorax pools of native mosquito species was 4.9% for Cx. pervigilans and 0% for Opifex fuscus. For the overall MIR, site and mosquito species were significant predictors of Plasmodium overall MIR. Aedes notoscriptus and Cx. pervigilans were positive for malaria DNA in the thorax samples, indicating that they may play a role as avian malaria vectors. Four different Plasmodium lineages (SYAT05, LINN1, GRW6, and a new lineage of P (Haemamoeba) sp. AENOT11) were identified in the pooled samples. CONCLUSIONS: This is the first detection of avian Plasmodium DNA extracted from thoraxes of native Culex and introduced Aedes mosquito species in New Zealand and therefore the first study providing an indication of potential vectors in this country.

Prevalence and genetic diversity of simian malaria in wild macaque populations across Thailand: Implications for human health.

Over the past year, P. falciparum infections have declined in Thailand, yet nonhuman primate malaria infections have correspondingly increased, including Plasmodium knowlesi and P. cynomolgi. Nevertheless, little is known about simian malaria in its natural macaque hosts, Macaca mulatta and Macaca fascicularis. This study aims to address several research questions, including the prevalence and distribution of simian malaria in these two Thai wild macaque species, variations in infection between different macaque species and between M. fascicularis subspecies, and the genetic composition of these pathogens. Blood samples were collected from 82 M. mulatta and 690 M. fascicularis across 15 locations in Thailand, as well as two locations in Vietnam and Myanmar. We employed quantitative real-time PCR targeting the Plasmodium genus-specific 18S ribosomal RNA (rRNA) gene to detect malaria infection, with a limit of detection set at 1,215.98 parasites per mL. We genotyped eight microsatellite markers, and the P. cynomolgi dihydrofolate reductase gene (DHFR) was sequenced (N = 29). In total, 100 of 772 samples (13 %) tested positive for malaria, including 45 (13 %) for P. cynomolgi, 37 (13 %) for P. inui, 16 (5 %) for P. coatneyi, and 2 (0.25 %) for Hepatocystis sp. in Saraburi, central and Ranong, southern Thailand. Notably, simian malaria infection was observed exclusively in M. fascicularis and not in M. mulatta (P = 0.0002). Particularly, P. cynomolgi was detected in 21.7 % (45/207) of M. f. fascicularis living in Wat Tham Phrapothisat, Saraburi Province. The infection with simian malaria was statistically different between M. fascicularis and M. mulatta (P = 0.0002) but not within M. fascicularis subspecies (P = 0.78). A haplotype network analysis revealed that P. cynomolgi shares a lineage with reference strains obtained from macaques. No mutation in the predicted binding pocket of PcyDHFR to pyrimethamine was observed. This study reveals a significant prevalence of simian malaria infection in M. fascicularis. The clonal genotypes of P. cynomolgi suggest in-reservoir breeding. These findings raise concerns about the potential spread of nonhuman primate malaria to humans and underscore the need for preventive measures.

Ready for renascence in mosquito: The regulation of gene expression in Plasmodium sexual development.

Malaria remains one of the most perilous vector-borne infectious diseases for humans globally. Sexual gametocyte represents the exclusive stage at which malaria parasites are transmitted from the vertebrate to the Anopheles host. The feasible and effective approach to prevent malaria transmission is by addressing the sexual developmental processes, that is, gametocytogenesis and gametogenesis. Thus, this review will comprehensively cover advances in the regulation of gene expression surrounding the transmissible stages, including epigenetic, transcriptional, and post-transcriptional control.

Dynamics of Plasmodium species and genotype diversity in adults with asymptomatic infection in Gabon.

OBJECTIVES: We investigated the diversity and dynamics of Plasmodium infection in serially collected samples from asymptomatic participants of a clinical trial assessing the efficacy and safety of ivermectin in Gabon. We checked whether the baseline sample reflected the P. falciparum genotype and Plasmodium species diversity seen over 7 days of follow-up. METHODS: Blood samples were collected at inclusion, every 8 hours until hour 72, daily until day 7, and on day 14. Plasmodium species was determined by qPCR and pfmsp1 length polymorphism was assessed for P. falciparum genotyping. RESULTS: In 17/48 (35%) individuals, all pfmsp1 genotypes identified during the assessed period were detected at baseline; in 31/48 (65%), new genotypes were found during follow-up. Additional sampling at hour 24 allowed the identification of all genotypes seen over 7 days in 50% of the individuals. Ivermectin did not impact the genotype dynamics. Mixed Plasmodium spp. infections were detected in 28/49 (57%) individuals at baseline, and detection of non-falciparum infections during follow-up varied. CONCLUSIONS: Our results reveal complex intra-host dynamics of P. falciparum genotypes and Plasmodium species and underscore the importance of serial sampling in clinical trials for antimalarial drugs with asymptomatically P. falciparum-infected individuals. This might allow a more accurate identification of genotypes in multiple infections, impacting the assessment of drug efficacy.

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

BACKGROUND: Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. METHODS: Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. RESULTS AND CONCLUSION: The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Ultrasensitive malaria detection system for Anopheles mosquito field surveillance using droplet digital PCR.

Malaria remains a significant global public health concern, with a recent increase in the number of zoonotic malaria cases in Southeast Asian countries. However, limited reports on the vector for zoonotic malaria exist owing to difficulties in detecting parasite DNA in Anopheles mosquito vectors. Herein, we demonstrate for the first time that several Anopheles mosquitoes contain simian malaria parasite DNA using droplet digital PCR (ddPCR), a highly sensitive PCR method. An entomological survey was conducted to identify simian malaria vector species at Phra Phothisat Temple (PPT), central Thailand, recognized for a high prevalence of simian malaria in wild cynomolgus macaques. A total of 152 mosquitoes from six anopheline species were collected and first analyzed by a standard 18S rRNA nested-PCR analysis for malaria parasite which yielded negative results in all collected mosquitoes. Later, ddPCR was used and could detect simian malaria parasite DNA, i.e. Plasmodium cynomolgi, in 25 collected mosquitoes. And this is the first report of simian malaria parasite DNA detection in Anopheles sawadwongporni. This finding proves that ddPCR is a powerful tool for detecting simian malarial parasite DNA in Anopheles mosquitoes and can expand our understanding of the zoonotic potential of malaria transmission between monkeys and humans.

RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

Contribution of Anopheles gambiae sensu lato mosquitoes to malaria transmission during the dry season in Djoumouna and Ntoula villages in the Republic of the Congo.

BACKGROUND: Mosquitoes belonging to the Anopheles gambiae sensu lato complex play a major role in malaria transmission across Africa. This study assessed the relative importance of members of An. gambiae s.l. in malaria transmission in two rural villages in the Republic of the Congo. METHODS: Adult mosquitoes were collected using electric aspirators from June to September 2022 in Djoumouna and Ntoula villages and were sorted by taxa based on their morphological features. Anopheles gambiae s.l. females were also molecularly identified. A TaqMan-based assay and a nested polymerase chain reaction (PCR) were performed to determine Plasmodium spp. in the mosquitoes. Entomological indexes were estimated, including man-biting rate, entomological inoculation rate (EIR), and diversity index. RESULTS: Among 176 mosquitoes collected, An. gambiae s.l. was predominant (85.8%), followed by Culex spp. (13.6%) and Aedes spp. (0.6%). Three members of the An. gambiae s.l. complex were collected in both villages, namely An. gambiae sensu stricto (74.3%), Anopheles coluzzii (22.9%) and Anopheles arabiensis (2.8%). Three Plasmodium species were detected in An. gambiae s.s. and An. coluzzii (Plasmodium falciparum, P. malariae and P. ovale), while only P. falciparum and P. malariae were found in An. arabiensis. In general, the Plasmodium infection rate was 35.1% (53/151) using the TaqMan-based assay, and nested PCR confirmed 77.4% (41/53) of those infections. The nightly EIR of An. gambiae s.l. was 0.125 infectious bites per person per night (ib/p/n) in Djoumouna and 0.08 ib/p/n in Ntoula. The EIR of An. gambiae s.s. in Djoumouna (0.11 ib/p/n) and Ntoula (0.04 ib/p/n) was higher than that of An. coluzzii (0.01 and 0.03 ib/p/n) and An. arabiensis (0.005 and 0.0 ib/p/n). CONCLUSIONS: This study provides baseline information on the dominant vectors and dynamics of malaria transmission in the rural areas of the Republic of the Congo during the dry season. In the two sampled villages, An. gambiae s.s. appears to play a predominant role in Plasmodium spp.

The Genome of Plasmodium gonderi: Insights into the Evolution of Human Malaria Parasites.

Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium.

Itaconate impairs immune control of Plasmodium by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells.

Severe forms of malaria are associated with systemic inflammation and host metabolism disorders; however, the interplay between these outcomes is poorly understood. Using a Plasmodium chabaudi model of malaria, we demonstrate that interferon (IFN) γ boosts glycolysis in splenic monocyte-derived dendritic cells (MODCs), leading to itaconate accumulation and disruption in the TCA cycle. Increased itaconate levels reduce mitochondrial functionality, which associates with organellar nucleic acid release and MODC restraint. We hypothesize that dysfunctional mitochondria release degraded DNA into the cytosol. Once mitochondrial DNA is sensitized, the activation of IRF3 and IRF7 promotes the expression of IFN-stimulated genes and checkpoint markers. Indeed, depletion of the STING-IRF3/IRF7 axis reduces PD-L1 expression, enabling activation of CD8+ T cells that control parasite proliferation. In summary, mitochondrial disruption caused by itaconate in MODCs leads to a suppressive effect in CD8+ T cells, which enhances parasitemia. We provide evidence that ACOD1 and itaconate are potential targets for adjunct antimalarial therapy.

Proteome-Wide Identification of RNA-dependent proteins and an emerging role for RNAs in Plasmodium falciparum protein complexes.

Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.

Evaluation of the Performance of Rapid Diagnostic Tests for Malaria Diagnosis and Mapping of Different Plasmodium Species in Mali.

BACKGROUND: The first-line diagnosis of malaria in Mali is based on the use of rapid diagnostic tests (RDT) that detect the Histidin Rich Protein 2 (HRP2) antigen specific to Plasmodium falciparum. Our study, based on a real-time polymerase chain reaction (qPCR) gold standard, aimed to describe the distribution of the Plasmodium species in each administrative region of Mali and to assess the performance of RDTs. METHODS: We randomly selected 150 malaria-negative and up to 30 malaria-positive RDTs in 41 sites distributed in 9 regions of Mali. DNA extracted from the RDT nitrocellulose strip was assayed with a pan-Plasmodium qPCR. Positive samples were then analyzed with P. falciparum-, P. malariae-, P. vivax-, or P. ovale-specific qPCRs. RESULTS: Of the 1496 RDTs, 258 (18.6%) were positive for Plasmodium spp., of which 96.9% were P. falciparum. The P. vivax prevalence reached 21.1% in the north. RDT displayed acceptable diagnostic indices; the lower CI95% bounds of Youden indices were all ≥0.50, except in the north (Youden index 0.66 (95% CI [0.44-0.82]) and 0.63 (95% CI [0.33-0.83]. CONCLUSIONS: Overall, RDT diagnostic indices are adequate for the biological diagnosis of malaria in Mali. We recommend the use of RDTs detecting P. vivax-specific antigens in the north.

Assessing the daily natural history of asymptomatic Plasmodium infections in adults and older children in Katakwi, Uganda: a longitudinal cohort study.

BACKGROUND: Low-density asymptomatic Plasmodium infections are prevalent in endemic areas, but little is known about their natural history. The trajectories of these infections and their propensity to fluctuate to undetectable densities can affect detection in clinical trials and field studies. We aimed to classify the natural history of these infections in a high transmission area over 29 days. METHODS: In this longitudinal cohort study, we enrolled healthy, malaria-asymptomatic, afebrile, adults (age 18-59 years) and older children (age 8-17 years) in Katakwi District, Uganda, who were negative for Plasmodium infection on rapid diagnostic tests. Participants were instructed to self-collect one dried blood spot (DBS) per day for a maximum of 29 days. We excluded people if they were pregnant or taking antimalarials. During weekly clinic visits, staff collected a DBS and a 4 mL sample of venous blood. We analysed DBSs by Plasmodium 18S rRNA quantitative RT-PCR (qRT-PCR). We classified DBS by infection type as negative, P falciparum, non-P falciparum, or mixed. We plotted infection type over time for each participant and categorised trajectories as negative, new, cleared, chronic, or indeterminate infections. To estimate the effect of single timepoint sampling, we calculated the daily prevalence for each study day and estimated the number of infections that would have been detected in our population if sampling frequency was reduced. FINDINGS: Between April 9 and May 20, 2021, 3577 DBSs were collected by 128 (40 male adults, 60 female adults, 12 male children, and 16 female children) study participants. 2287 (64%) DBSs were categorised as negative, 751 (21%) as positive for P falciparum, 507 (14%) as positive for non-P falciparum, and 32 (1%) as mixed infections. Daily Plasmodium prevalence in the population ranged from 45·3% (95% CI 36·6-54·1) at baseline to 30·3% (21·9-38·6) on day 24. 37 (95%) of 39 P falciparum and 35 (85%) of 41 non-P falciparum infections would have been detected with every other day sampling, whereas, with weekly sampling, 35 (90%) P falciparum infections and 31 (76%) non-P falciparum infections would have been detected. INTERPRETATION: Parasite dynamics and species are highly variable among low-density asymptomatic Plasmodium infections. Sampling every other day or every 3 days detected a similar proportion of infections as daily sampling, whereas testing once per week or even less frequently could misclassify up to a third of the infections. Even using highly sensitive diagnostics, single timepoint testing might misclassify the true infection status of an individual. FUNDING: US National Institutes of Health and Bill and Melinda Gates Foundation.