Molecular Epidemiology of Plasmodium species in Conflicted Federally Administered Tribal Area (FATA) Pakistan

Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistan’s war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria-related education locally, targeting children and parents alike.

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.

Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda

Background The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. Methods The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. Results Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months–81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. Conclusion The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

Anti-malarial plants in Ethiopia and their activities on drug resistant malaria

In Ethiopia, the impacts of malaria continue to cause a many number of morbidity and mortality that accounts to most outpatient observations. Ethiopia recently designed to attain nationwide malaria control by 2030 by beginning sub-national elimination in districts with low malaria transmission. However, the rises of drug-resistant parasites, especially Plasmodium falciparum hinder the malaria containment strategies. Plasmodium falciparum and Plasmodium vivax, dispersed all over the Ethiopia and accounting for 60% and 40% of malaria cases respectively. The aim of this report was to overview the phytochemical constituents, diversity and effect of some compound extracts on drug resistant plasmodium species. Many plant species, a total 200 identified by 82 studies, are used in traditional malaria treatments throughout the country. Allium sativum, Croton macrostachyus and Carica papaya were the more frequently used medicinal plants species. There are so many phytochemicals constituents found in medicinal plants used to treat malaria. Alkaloids, Flavonoids, Phenolics, Terpenoid and Glycosides are the most reported for their effective activity on drug resistant malaria.

Population-level genome-wide STR discovery and validation for population structure and genetic diversity assessment of Plasmodium species

Short tandem repeats (STRs) are highly informative genetic markers that have been used extensively in population genetics analysis. They are an important source of genetic diversity and can also have functional impact. Despite the availability of bioinformatic methods that permit large-scale genome-wide genotyping of STRs from whole genome sequencing data, they have not previously been applied to sequencing data from large collections of malaria parasite field samples. Here, we have genotyped STRs using HipSTR in more than 3,000 Plasmodium falciparum and 174 Plasmodium vivax published whole-genome sequence data from samples collected across the globe. High levels of noise and variability in the resultant callset necessitated the development of a novel method for quality control of STR genotype calls. A set of high-quality STR loci (6,768 from P. falciparum and 3,496 from P. vivax) were used to study Plasmodium genetic diversity, population structures and genomic signatures of selection and these were compared to genome-wide single nucleotide polymorphism (SNP) genotyping data. In addition, the genome-wide information about genetic variation and other characteristics of STRs in P. falciparum and P. vivax have been available in an interactive web-based R Shiny application PlasmoSTR (https://github.com/bahlolab/PlasmoSTR).

Structural activity relationship of metallo-aminoquines as a next generation antimalarials

Apicomplexian parasite of the genus Plasmodium is the causative agent of malaria, one of the most devastating, furious and common infectious disease throughout the world. According to the latest World malaria report, there were 229 million cases of malaria in 2019 majorly consisting of children under 5 years of age. Some of known analogues viz. quinine, quinoline-containing compounds have been used for last century in the clinical treatment of malaria. Past few decades have witnessed the emergence of multi-drug resistance (MDR) strains of Plasmodium species to existing antimalarials pressing the need for new drug candidates. For the past few decades bioorganometallic approach to malaria therapy has been introduced which led to the discovery of noval metalcontaining aminoquinolines analogues viz. ferroquine (FQ or 1), Ruthenoquine (RQ or 2) and other related potent metal-analogues. It observed that some metal containing analogues (Fe-, Rh-, Ru-, Re-, Au-, Zn-, Cr-, Pd-, Sn-, Cd-, Ir-, Co-, Cu-, and Mn-aminoquines) were more potent; however, some were equally potent as Chloroquine (CQ) and 1. This is probably due to the intertion of metals in the CQ via various approaches, which might be a very attractive strategy to develop a SAR of novel metal containing antimalarials. Thus, this review aims to summarize the SAR of metal containing aminoquines towards the discovery of potent antimalarial hybrids to provide an insight for rational designs of more effective and less toxic metal containing amoniquines.

Synthesis and in vitro drug release of primaquine phosphate loaded PLGA nanoparticles

Plasmodium falciparum is one of the most common resistant Plasmodium species responsible for high rates of morbidity and mortality in malaria patients. Clinical guidelines for the management of Plasmodium falciparum include the use of a dose of primaquine phosphate resulting intolerable side effects. Therefore, the aim of this work was to formulate primaquine phosphate-loaded PLGA nanoparticles by using a nanoprecipitation method in order to increase its bioavailability to minimize drug intake. This leads to reduced toxicity and better therapeutic efficacy of the drug. The synthesized nanoparticles were characterized by using dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transformed infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and powder X-ray diffraction (XRD). TEM analysis revealed the presence of smooth spherical-shaped nanoparticles. The drug DLS analysis confirmed the presence of negatively charged nanoparticles with particle size in the range of 100-400 nm. The drug release study was performed to analyses different kinetic models like zero-order model, first-order model, Higuchi model, Hixson-Crowell model, and Korsmeyer-Peppas model.

Structure-Based Virtual Screening, Insilico Docking, ADME Prediction, and Molecular Dynamics Studies for Identification of Potential NMT Inhibitors Against Malaria

One of the major public health problems globally, malaria, is mainly caused protozoan parasites from the genus Plasmodium, and commonly spreads to people through the bites of infected female mosquitoes of the genus Anopheles. Strategies for treatment, prevention, and control are available for malaria but the eradication of malaria still poses great challenge due to plasmodium’s drug resistance over the past decades. Development of novel antimalarial drugs remains a significant task to protect people from malaria. N-Myristoyl transferase is responsible for the N-Myristoylation catalysis process and the survival of Plasmodium species. Thus, it is considered a therapeutic drug target in protozoans and was recently validated as a significant target for Plasmodium vivax. In this present scenario, we endeavour to identify effective NMT inhibitors to prevent the onset of malaria in the human species. Initially, the structure-based virtual screening was executed against ZINC database and four potential candidates for NMT were identified. Furthermore, the four identified compounds were subjected to ADME prediction and all the four compounds found within adequate range with predicted ADME properties. Eventually, we conducted the molecular dynamics simulation to investigate the binding stability of top three protein-ligand complexes at different time scale by employing the tool Desmond. The molecular dynamics simulation studies revealed the protein-ligand complexes were stable throughout the entire simulation. Besides, we noticed that the residues ASN 365, PHE 103 and HIS 213 of NMT were crucially involved in the formation of various intermolecular interactions, significantly contributing to the stability of protein-ligand complexes. From this computational investigation, we suggest that the three identified potential compounds are extremely useful for further lead optimization and drug development.

Fast Detection And Quantification of Plasmodium Species Infected Erythrocytes In A Non-Endemic Region By Using The Sysmex XN-31 Analyzer

BACKGOUND: Due to increased travel from endemic countries, malaria occurs more frequently in non-endemic regions. It is a challenge for diagnostic laboratories in non-endemic countries to provide reliable results, as experience of staff is often limited to only a few cases per year. In this study, we evaluated the diagnostic accuracy of the fully automated Sysmex XN-31 malaria analyzer in a routine diagnostic setting in a non-endemic region. METHODS: Samples from 112 patients suspected for malaria were examined by the Sysmex XN-31 analyzer to determine the absolute count of malaria-infected red blood cells count (MI-RBC/µl). Microscopic examination of both Quantitative Blood Coat capillary tubes and thick and thin blood films were used as reference methods. Limits of blank (LoB), detection (LoD) and quantification (LoQ) were investigated using an in vitro Plasmodium falciparum culture . Nine hundred twenty samples of patients with RBC abnormalities were included to determine which RBC abnormalities trigger indeterminate or false positive results. RESULTS: No false positive nor false negative results were obtained for the examined patient samples suspected for malaria. For 3% of samples an indeterminate result by the XN-31 was obtained. The Passing-Bablok regression line for diagnostic accuracy of the parasitemia was y = 39.75 + 0.7892x showing a positive bias of about 21% when comparing the MI-RBC results to microscopy. The LoB, LoD and LoQ were calculated to be 4.7, 5.9, and 19.0 infected RBC/mL, respectively. From the 920 abnormal RBC samples collected, 4.6% resulted in a false positive MI-RBC result and almost half of the samples produced indeterminate results. These results were related to increases in nucleated red blood cells, reticulocytes and other abnormal RBC morphologies such as sickle cells. CONCLUSIONS: Based on the results we conclude that the XN-31 is a fast and reliable screening method in the detection and quantification of Plasmodium species in patients However, if an abnormal red blood cell morphology is present, the results of the XN-31 should be interpreted with caution as false positive results can be caused by interfering abnormal erythrocytes.