Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4

Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.

Bagaza virus and Plasmodium spp. coinfection in red-legged partridges ( Alectoris rufa ), in southern Spain 2019

Flaviviruses West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) virus and avian malaria parasites are vector borne pathogens that circulate naturally between avian and mosquito hosts. WNV and USUV and potentially also BAGV constitute zoonoses. Temporal and spatial co-circulation and co-infection with Plasmodium spp., and West Nile virus has been documented in birds and mosquito vectors, and fatally USUV infected passerines coinfected with Plasmodium spp. had more severe lesions. Also, WNV, USUV and BAGV have been found to co-circulate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or co-infections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain due to coinfection with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where co-circulation of BAGV, USUV and WNV was confirmed in 2011 and 2013. Partridges were found dead in early October 2019. Birds had mottled locally pale pectoral muscles, enlarged, congestive greenish-black tinged livers and enlarged kidneys. Microscopically congestion and predominantly mononuclear inflammatory infiltrates were evident and Plasmodium phanerozoites were present in the liver, spleen, kidneys, muscle and skin. Molecular testing and sequencing detected Plasmodium spp. and BAGV in different tissues of the partridges, and immunohistochemistry confirmed the presence and co-localization of both pathogens in the liver and spleen. Although Plasmodium spp. are known to be highly prevalent in red-legged partridges, this is the first account of mortality caused by co-infection with BAGV and Plasmodium sp. Due to the importance of the red-legged partridge in the ecosystem of the Iberian Peninsula and as driver of regional economy such mortalities are of concern. Also, they may reflect climate change related changes in host, vector and pathogen ecology and interactions that could emerge similarly in other pathogens.

Impact of Drug Pressure versus Limited Access to Drug in Malaria Control: The Dilemma

Malaria burden has severe impact on the world. Several arsenals, including the use of antimalarials, are in place to curb the malaria burden. However, the application of these antimalarials has two extremes, limited access to drug and drug pressure, which may have similar impact on malaria control, leading to treatment failure through divergent mechanisms. Limited access to drugs ensures that patients do not get the right doses of the antimalarials in order to have an effective plasma concentration to kill the malaria parasites, which leads to treatment failure and overall reduction in malaria control via increased transmission rate. On the other hand, drug pressure can lead to the selection of drug resistance phenotypes in a subpopulation of the malaria parasites as they mutate in order to adapt. This also leads to a reduction in malaria control. Addressing these extremes in antimalarial application can be essential in maintaining the relevance of the conventional antimalarials in winning the war against malaria.

Survey on Avian Malaria Parasites in Village Chickens (Gallus gallus domesticus) in Gombe Local Government Area, Gombe State, Nigeria

Reports of avian malaria parasites in village chicken in Nigeria generally remain fragmentary and scarce. The study was conducted in Gombe Local Government Area of Gombe State, Nigeria to investigate avian malaria parasites in Village Chickens (Gallus gallus domesticus) and to determine the risk factors associated with the prevalence of the haemoparasites. A total of 530 village chickens blood samples were obtained from apparently healthy village chickens' brachial veins using sterile 2mls syringes and 23 gauge needles. Thin blood smear was made from each blood sample, and Giemsa stained and examined for the presence of avian haemoparasites under an electro-microscope. The result indicates 23.8% overall prevalence rate of three species of avian malaria parasites consisting of Plasmodium, Haemoproteus and Leucocytozoon species.Plasmodium spp. has the highest prevalent rate of 13.0% followed by Haemoproteus spp. (5.1%), mixed Plasmodium spp. + Haemoproteus spp. (4.9%) infection and Leucocytozoon spp. (0.8%). Prevalence of avian malaria parasites was significantly higher in cocks compared to hens (p < 0.05), as well as higher in adults compared to growers chickens (p < 0.05). This study also showed a higher prevalence of avian malaria parasites during the rainy season compared to the dry season of the study period. It was concluded that haemoparasites of Plasmodium, Haemoproteus and Leucocytozoon species that occur in both single and mixed infections are prevalent among village chickens that are apparently healthy in Gombe Local Government Area of Gombe State, Nigeria.

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This is a syncytial mode of replication involving asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite replication is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear replications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.

A Mitochondria-Penetrating Peptide Exerts Potent Anti-Plasmodium Activity and Localizes at Parasites’ Mitochondria

Mitochondria are considered a novel drug target as they play a key role in energy production and programmed cell death of eukaryotic cells. The mitochondria of malaria parasites differ from those of their vertebrate hosts, contributing to the drug selectivity and the development of antimalarial drugs. (Fxr)3, a mitochondria-penetrating peptide or MPP, entered malaria-infected red cells without disrupting the membrane and subsequently killed the blood stage of P. falciparum parasites. The effects were more potent on the late stages than on the younger stages. Confocal microscopy showed that the (Fxr)3 intensely localized at the parasite mitochondria. (Fxr)3 highly affected both the lab-strain, chloroquine-resistant K1, and freshly isolated malaria parasites. (Fxr)3 (1 ng/mL to 10 μg/mL) was rarely toxic towards various mammalian cells, i.e., mouse fibroblasts (L929), human leukocytes and erythrocytes. At a thousand times higher concentration (100 μg/mL) than that of the antimalarial activity, cytotoxicity and hemolytic activity of (Fxr)3 were observed. Compared with the known antimalarial drug, atovaquone, (Fxr)3 exhibited more rapid killing activity. This is the first report on antimalarial activity of (Fxr)3, showing localization at parasites’ mitochondria.

Prevalence of Infection and Malaria Parasite Density among Under Five Children: A Case Study of Dunukofia Rural Community in Anambra State, Nigeria

Aim: Malaria still remains an overwhelming cause of morbidity and mortality among children under five years of age, especially in sub-Saharan Africa. The study was carried out to evaluate malaria prevalence amongst children less than five years old. Study Design: A cross sectional study was carried out. The study adopted a retrospective descriptive survey using the hospital records and diagnostic cross sectional survey by examination of blood samples across three variables: gender, age group and mosquito net usage. Duration: The study was done in 2021 from the month of March to April in the rural community. Methodology: Parasitological diagnosis was with Plasmodium falciparum histidine-rich protein 2-based malaria Rapid Diagnostic Test (RDT) and microscopy of giemsa-stained blood smears. Demographic information was collected using questionnaire. Results: Three hundred (300) children aged less than five years malaria infection status was investigate, 174 (58.00% ) of them were females while 126 (42.00%. ) were males. Twenty one percent (21.00%) of the respondents are <1 year, 23.33% (70) of them are between the ages of 2 to 3 years, while 55.67% (167) were 4 years and above. Current malaria prevalence was higher with microscopy (67.33%) than that of RDT (23.33%). Also, previous RDT results showed that there was a higher prevalence (73.56%) of malaria parasites in females than males (58.73%). The microscopy results showed that males had a higher prevalence (38.10%) of malaria parasites than females (12.64%). Overall gender result also revealed that males had a higher prevalence (96.83%) of malaria parasites than females (86.21%). There was a significant difference in the prevalence result with gender (P<0.05). Females had higher parasite density (28.05±15.390) than males (23.22±19.171), there was no significant difference (P>0.05). It further revealed that children from 4 years and above had higher intensity (29.68±17.357) while those of 1 year and below had the least (14.89±16.069). However, there was no significant difference in the malaria parasite among the age groups of patients (P>0.05). Conclusion: Prevalence of malaria parasitaemia was still high in Dunukaofia, Anamba State, Nigeria despite various control measures and interventions put in place by WHO.

Plasmodium falciparum and Plasmodium vivax Adjust Investment in Transmission in Response to Change in Transmission Intensity: A Review of the Current State of Research

Malaria parasites can adjust the proportion of parasites that develop into gametocytes, and thus the probability for human-to-vector transmission, through changes in the gametocyte conversion rate. Understanding the factors that impact the commitment of malaria parasites to transmission is required to design better control interventions. Plasmodium spp. persist across countries with vast differences in transmission intensities, and in sites where transmission is highly seasonal. Mounting evidence shows that Plasmodium spp. adjusts the investment in transmission according to seasonality of vector abundance, and transmission intensity. Various techniques to determine the investment in transmission are available, i.e., short-term culture, where the conversion rate can be measured most directly, genome and transcriptome studies, quantification of mature gametocytes, and mosquito feeding assays. In sites with seasonal transmission, the proportion of gametocytes, their densities and infectivity are higher during the wet season, when vectors are plentiful. When countries with pronounced differences in transmission intensity were compared, the investment in transmission was higher when transmission was low, thus maximizing the parasite’s chances to be transmitted to mosquitoes. Increased transmissibility of residual infections after a successful reduction of malaria transmission levels need to be considered when designing intervention measures.