Preliminary insight on diarylpentanoids as potential antimalarials: In silico, in vitro pLDH and in vivo zebrafish toxicity assessment.

Malaria remains a major public health problem worldwide, including in Southeast Asia. Chemotherapeutic agents such as chloroquine (CQ) are effective, but problems with drug resistance and toxicity have necessitated a continuous search for new effective antimalarial agents. Here we report on a virtual screening of ∼300 diarylpentanoids and derivatives, in search of potential Plasmodium falciparum lactate dehydrogenase (PfLDH) inhibitors with acceptable drug-like properties. Several molecules with binding affinities comparable to CQ were chosen for in vitro validation of antimalarial efficacy. Among them, MS33A, MS33C and MS34C are the most promising against CQ-sensitive (3D7) with EC50 values of 1.6, 2.5 and 3.1 µM, respectively. Meanwhile, MS87 (EC50 of 1.85 µM) shown the most active against the CQ-resistant Gombak A strain, and MS33A and MS33C the most effective P. knowlesi inhibitors (EC50 of 3.6 and 5.1 µM, respectively). The in vitro cytotoxicity of selected diarylpentanoids (MS33A, MS33C, MS34C and MS87) was tested on Vero mammalian cells to evaluate parasite selectivity (SI), showing moderate to low cytotoxicity (CC50 > 82 µM). In addition, MS87 exhibited a high SI and the lowest resistance index (RI), suggesting that MS87 may exert effective parasite inhibition with low resistance potential in the CQ-resistant P. falciparum strain. Furthermore, the in vivo toxicity of the molecules on early embryonic development, the cardiovascular system, heart rate, motor activity and apoptosis were assessed in a zebrafish animal model. The overall results indicate the preliminary potential of diarylpentanoids, which need further investigation for their development as new antimalarial agents.

The prevalence of simian malaria in wild long-tailed macaques throughout Peninsular Malaysia.

The parasite Plasmodium knowlesi has been the sole cause of malaria in Malaysia from 2018 to 2022. The persistence of this zoonotic species has hampered Malaysia's progress towards achieving the malaria-free status awarded by the World Health Organisation (WHO). Due to the zoonotic nature of P. knowlesi infections, it is important to study the prevalence of the parasite in the macaque host, the long-tailed macaque (Macaca fascicularis). Apart from P. knowlesi, the long-tailed macaque is also able to harbour Plasmodium cynomolgi, Plasmodium inui, Plasmodium caotneyi and Plasmodium fieldi. Here we report the prevalence of the 5 simian malaria parasites in the wild long-tailed macaque population in 12 out of the 13 states in Peninsular Malaysia using a nested PCR approach targeting the 18s ribosomal RNA (18s rRNA) gene. It was found that all five Plasmodium species were widely distributed throughout Peninsular Malaysia except for states with major cities such as Kuala Lumpur and Putrajaya. Of note, Pahang reported a malaria prevalence of 100% in the long-tailed macaque population, identifying it as a potential hotspot for zoonotic transmission. Overall, this study shows the distribution of the 5 simian malaria parasite species throughout Peninsular Malaysia, the data of which could be used to guide future malaria control interventions to target zoonotic malaria.

Synthesis and biological evaluation of hydantoin derivatives as potent antiplasmodial agents.

Malaria, a devastating disease, has claimed numerous lives and caused considerable suffering, with young children and pregnant women being the most severely affected group. However, the emergence of multidrug-resistant strains of Plasmodium and the adverse side effects associated with existing antimalarial drugs underscore the urgent need for the development of novel, well-tolerated, and more efficient drugs to combat this global health threat. To address these challenges, six new hydantoins derivatives were synthesized and evaluated for their in vitro antiplasmodial activity. Notably, compound 2c exhibited excellent inhibitory activity against the tested Pf3D7 strain, with an IC50 value of 3.97 ± 0.01 nM, three-fold better than chloroquine. Following closely, compound 3b demonstrated an IC50 value of 27.52 ± 3.37 µM against the Pf3D7 strain in vitro. Additionally, all the hydantoins derivatives tested showed inactive against human MCR-5 cells, with an IC50 value exceeding 100 µM. In summary, the hydantoin derivative 2c emerges as a promising candidate for further exploration as an antiplasmodial compound.

Characterization of merozoite-specific thrombospondin-related anonymous protein (MTRAP) in Plasmodium vivax and P. knowlesi parasites.

Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.

Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.

Digital microscope-assisted photography improves the accuracy of mosquito wing measurement.

Wing measurement is an important parameter in many entomological studies. However, the methods of measuring wings vary with studies, and a gold standard method was not available for this procedure. This in turn limits researchers from confidently comparing their research findings with published data collected by other means of measurement. This study investigated the interchangeability of three commonly available methods for wing measurement, namely the calliper method, stereomicroscope-assisted photography method, and digital microscope-assisted photography method, using the laboratory colony of Aedes aegypti. It was found that the calliper method and the photography-based methods yielded similar results, hence the good interchangeability of these methods. Nevertheless, the digital microscope-assisted photography method yielded more accurate measurements, due to the higher resolution of the captured photos, and minimal technical bias during the data collection, as compared to the calliper-based and stereomicroscope-assisted photography methods. This study served as a reference for researchers to select the most suitable measurement method in future studies.

Porous borders at the wild-crop interface promote weed adaptation in Southeast Asia.

High reproductive compatibility between crops and their wild relatives can provide benefits for crop breeding but also poses risks for agricultural weed evolution. Weedy rice is a feral relative of rice that infests paddies and causes severe crop losses worldwide. In regions of tropical Asia where the wild progenitor of rice occurs, weedy rice could be influenced by hybridization with the wild species. Genomic analysis of this phenomenon has been very limited. Here we use whole genome sequence analyses of 217 wild, weedy and cultivated rice samples to show that wild rice hybridization has contributed substantially to the evolution of Southeast Asian weedy rice, with some strains acquiring weed-adaptive traits through introgression from the wild progenitor. Our study highlights how adaptive introgression from wild species can contribute to agricultural weed evolution, and it provides a case study of parallel evolution of weediness in independently-evolved strains of a weedy crop relative.

A visualized hybrid PCR-LAMP assay for the detection of human Plasmodium species.

Combining the advantages of PCR and LAMP, we described a new technique, namely PCR-LAMP, for malaria diagnosis. The whole process of DNA amplification can be completed in 35 min. This hybrid amplification technique markedly improved the sensitivity of detection compared to the classic single PCR or LAMP assay alone. PCR-LAMP assay had a detection limit of 1 copy/µL for P. knowlesi and P. ovale, 0.1 copy/µL for P. vivax, P. falciparum and P. malariae, respectively. To facilitate the endpoint detection, xylenol orange was added. Positive samples were indicated in orange while negative reactions were violet. The inclusion of xylenol orange into the LAMP reaction mix significantly reduces the post-amplification workload. Without relying on the use of specific instruments, the color changes of the amplicons could be visualized directly through the naked eye. In conclusion, PCR-LAMP poses the potential to be developed as a new malaria molecular diagnosis tool.

Genetic polymorphism and clustering of the Plasmodium cynomolgi Duffy binding protein 1 region II of recent macaque isolates from Peninsular Malaysia.

Plasmodium cynomolgi is a simian malaria parasite that has been increasingly infecting humans. It is naturally present in the long-tailed and pig-tailed macaques in Southeast Asia. The P. cynomolgi Duffy binding protein 1 region II [PcDBP1(II)] plays an essential role in the invasion of the parasite into host erythrocytes. This study investigated the genetic polymorphism, natural selection and haplotype clustering of PcDBP1(II) from wild macaque isolates in Peninsular Malaysia. The genomic DNA of 50 P. cynomolgi isolates was extracted from the macaque blood samples. Their PcDBP1(II) gene was amplified using a semi-nested PCR, cloned into a plasmid vector and subsequently sequenced. The polymorphism, natural selection and haplotypes of PcDBP1(II) were analysed using MEGA X and DnaSP ver.6.12.03 programmes. The analyses revealed high genetic polymorphism of PcDBP1(II) (π = 0.026 ± 0.004; Hd = 0.996 ± 0.001), and it was under purifying (negative) selection. A total of 106 haplotypes of PcDBP1(II) were identified. Phylogenetic and haplotype analyses revealed two groups of PcDBP1(II). Amino acid length polymorphism was observed between the groups, which may lead to possible phenotypic difference between them.

Intestinal parasitic infections and risk analysis among urban refugees in the Klang Valley, Malaysia.

BACKGROUND: Global studies show intestinal parasitic infections (IPIs) have been introduced and spread with refugee inflows from low to high socio-economic countries. However, there is relatively limited information on the prevalence of infections among the community. METHODS: A 2-year cross-sectional study was conducted to determine the prevalence and associated risk factors for infections among urban refugees in the Klang Valley, Malaysia. A total of 418 faecal samples were collected and examined by microscopy. RESULTS: Faecal screening revealed moderate levels (32.3%) of infections in the community. Three nematode (Ascaris lumbricoides, Trichuris trichiura and hookworm) and three protozoan species (Entamoeba, Giardia and Cryptosporidium) were recorded, with the highest prevalence being A. lumbricoides (20.6%) followed by T. trichiura (10.3%), while other infections were <5%. Statistical analysis found that young males with less education were more likely to be infected with helminths. Additionally, living near waste disposal sites, the presence of stray animals, eating with bare hands, bare footedness, poor handwashing practices and no anthelmintic treatment constituted significant risk factors for helminth infections. Protozoan infections were linked to drinking tap water or from water dispensers and poor handwashing practices. CONCLUSIONS: These findings emphasize the importance of health education in addition to introduction of biannual anthelmintic treatment to promote community health and well-being.

Evaluation of A Simple DNA Extraction Method and Its Combination with Loop-Mediated Isothermal Amplification Assays for Rapid Plasmodium knowlesi Diagnosis.

The initial and vital stage in the diagnosis of malaria involves extracting DNA. The efficiency of malaria testing is restricted by the multiple steps involved in commercial DNA extraction kits. We attempted to improve an existing loop-mediated isothermal amplification (LAMP) for the detection of Plasmodium knowlesi by using a simple DNA extraction approach, making it a feasible option for mass screening. We utilized a simple nucleic acid extraction method directly from whole blood for the detection of P. knowlesi, taking only 5 min to complete. The extracted DNA was evaluated by two fluorescent-based LAMP and one colorimetric-based LAMP assay. The detection limit for both SYTO-LAMP and SYBR green-LAMP was 0.00001% and 0.0001% parasitemia, respectively. Meanwhile, neutral red-LAMP had a detection limit of 0.01% parasitemia. Combining this simple and inexpensive DNA extraction method, SYTO-LAMP could serve as an alternative molecular diagnosis for the detection of P. knowlesi and other human Plasmodium spp.

Genetic diversity and in silico analysis of Plasmodium knowlesi Serine Repeat Antigen (SERA) 3 antigen 2 in Malaysia.

Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic characterization on P. knowlesi SERA3 Ag2 (PkSERA3 Ag2) is lacking. In the present study, nucleotide diversity, natural selection, and haplotypes of PkSERA3 Ag2 in clinical samples from Peninsular Malaysia and Malaysian Borneo were investigated. A total of 50 P. knowlesi clinical samples were collected from Peninsular Malaysia and Malaysian Borneo. The PkSERA3 Ag2 gene was amplified using PCR, and subsequently cloned and sequenced. Genetic diversity, haplotype, natural selection as well as genetic structure and differentiation of PkSERA3 Ag2 were analysed. In addition, in silico analyses were performed to identify repeat motifs, B-cell epitopes, and antigenicity indices of the protein. Analysis of 114 PkSERA3 Ag2 sequences revealed high nucleotide diversity of the gene in Malaysia. A codon-based Z-test indicated that the gene underwent purifying selection. Haplotype and population structure analyses identified two distinct PkSERA3 Ag2 clusters (K = 2, ΔK = 721.14) but no clear genetic distinction between PkSERA3 Ag2 from Peninsular Malaysia and Malaysian Borneo. FST index indicated moderate differentiation of the gene. In silico analyses revealed unique repeat motifs among PkSERA3 Ag2 isolates. Moreover, the amino acid sequence of PkSERA3 Ag2 exhibited potential B-cell epitopes and possessed high antigenicity indices. These findings enhance the understanding of PkSERA3 Ag2 gene as well as its antigenic properties. Further validation is necessary to ascertain the utility of PkSERA3 Ag2 as a serological marker for P. knowlesi infection.