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Malaria, an ancient mosquito-borne illness caused by Plasmodium parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the P . falciparum Kelch 13 ( PfK13 ) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate PfK13 recombinant proteins incorporating of two specific SNPs mutations, PfK13 -V494I and PfK13 -N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of PfK13 mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between PfK13 -V494I and PfK13 -N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg/ml for PfK13 -WT, 0.18 mg/ml for PfK13 -V494I, and 0.28 mg/ml for PfK13 -N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants PfK13 -N537I (83 a.u.) and PfK13 -V494I (143 a.u.) compared to PfK13 -WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between PfK13 protein mutants with ART. This shows that the PfK13 mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R.

Malaysia successfully achieved zero indigenous human malaria cases since 2018. However, challenges persist from Plasmodium knowlesi (zoonotic malaria) and low-density infections, posing reintroduction risks in previously malaria-free areas. Addressing these hidden infections is critical for sustaining Malaysia’s elimination gains. This study investigated the persistence of low-density malaria transmission in high-risk localities declared malaria-free for at least three consecutive years. A community-based cross-sectional survey was conducted from June to October 2020 in 23 high-receptivity localities across Sabah, Perak, Kelantan, and Johor. Blood samples from asymptomatic residents were screened via conventional microscopy and nested PCR (nPCR) targeting the Plasmodium 18S rRNA gene, with positive nPCR products species-determined. Sociodemographic and geospatial data were analyzed for associations with infection status. Of 3,322 asymptomatic individuals, no infections were detected by microscopy, whereas nPCR revealed a low-density malaria prevalence of 1.86% (62/3,322). Infections comprised P. malariae (40.3%), P. vivax (29.0%), P. knowlesi (24.2%), P. falciparum (1.6%), P. cynomolgi (1.6%), and mixed P. vivax/P. knowlesi (3.2%). All PCR-positive cases originated from Sabah and an Orang Asli settlement in Perak. Adults (≥17 years) constituted the majority (~68%), with no significant difference in prevalence by gender or previous malaria history (p > 0.05). Asymptomatic low-density malaria infections persist in purportedly malaria-free communities, remaining undetectable by routine microscopy. These hidden parasite reservoirs pose a risk for malaria reintroduction, especially in receptive areas. Malaria surveillance programs must thus incorporate highly sensitive diagnostic tools to detect low-density infections and safeguard elimination gains. Intensified, targeted interventions in identified “malaria hotspots”, including community engagement and vector control, are crucial to eliminate residual foci and prevent disease resurgence.

Background Malaysia has declared its aim to eliminate malaria with a goal of achieving zero local transmission by the year 2020. However, targeting the human reservoir of infection, including those with asymptomatic infection is required to achieve malaria elimination. Diagnosing asymptomatic malaria is not as straightforward due to the obvious lack of clinical manifestations and often subpatent level of parasites. Accurate diagnosis of malaria is important for providing realistic estimates of malaria burden and preventing misinformed interventions. Low levels of parasitaemia acts as silent reservoir of transmission thus remains infectious to susceptible mosquito vectors. Hence, the aim of this study is to investigate the prevalence of asymptomatic submicroscopic malaria (SMM) in the District of Belaga, Sarawak. Methods In 2013, a total of 1744 dried blood spots (DBS) were obtained from residents of 8 longhouses who appeared healthy. Subsequently, 251 venous blood samples were collected from residents of 2 localities in 2014 based on the highest number of submicroscopic cases from prior findings. Thin and thick blood films were prepared from blood obtained from all participants in this study. Microscopic examination were carried out on all samples and a nested and nested multiplex PCR were performed on samples collected in 2013 and 2014 respectively. Results No malaria parasites were detected in all the Giemsa-stained blood films. However, of the 1744 samples, 29 (1.7%) were positive for Plasmodium vivax by PCR. Additionally, of the 251 samples, the most prevalent mono-infection detected by PCR was Plasmodium falciparum 50 (20%), followed by P. vivax 39 (16%), P. knowlesi 9 (4%), and mixed infections 20 (8%). Conclusions This research findings conclude evidence of Plasmodium by PCR, among samples previously undetectable by routine blood film microscopic examination, in local ethnic minority who are clinically healthy. SMM in Belaga district is attributed not only to P. vivax , but also to P. falciparum and P. knowlesi. In complementing efforts of programme managers, there is a need to increase surveillance for SMM nationwide to estimate the degree of SMM that warrant measures to block new transmission of malaria.

Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system.

Background The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum , the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum . Methods Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. Results The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. Conclusions The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

Objective Main malaria diagnosis is based on microscopic examination combined with rapid diagnostic tests. Both methods have low sensitivity and specificity. Loop-mediated isothermal amplification techniques have shown a sensitivity similar to PCR but with lower times of performance. This study aimed to assess a commercial LAMP for the diagnosis of malaria (Alethia ® Malaria) against the Nested-Multiplex-Malaria PCR, including the analytical sensitivity and the operational characteristics. Results One hundred five samples out of 114 rendered valid results, obtaining 85 positive samples and 18 negative samples with an agreement of 98% compared to the reference method with a sensitivity, specificity and kappa coefficient of 98.84%, 94.74% and 0.94 respectively, with only two discrepant samples. The turnaround time was estimated in 1 h and 30 min, with a cost of 32.67€ per determination. The results showed several advantages of the Alethia ® Malaria, as it was easy to perform, minimal training requirement and 40 min run. Moreover, it includes an internal control to avoid false negatives. However, it also showed some limitations such as the need for a specific amplification and detection device, the detection of only Plasmodium spp. and a very high price.

The risk of non-human primate (NHP) malaria transmission to humans is increasing, with Plasmodium knowlesi and Plasmodium cynomolgi emerging as significant zoonotic threats, particularly in Malaysia. While P. knowlesi is well-documented, P. cynomolgi infections in humans remain underreported, largely due to diagnostic challenges. Routine microscopy and standard molecular diagnostic tools often misdiagnose P. cynomolgi infections as P. vivax due to morphological similarities and genetic homology. We report a new case of a human P. cynomolgi infection misdiagnosed as Plasmodium vivax in a 32-year-old male with no prior malaria history or travel to endemic countries. The initial diagnoses made by the microscopy and qPCR conducted by the Kota Bharu Public Health Laboratory in Kelantan identified the infection as P. vivax. However, cross-examination by the Institute for Medical Research (IMR) revealed the presence of mixed-species infection, prompting further analysis. The real-time PCR and sequencing performed at MAPELAB, Spain, confirmed the co-infection of P. vivax and P. cynomolgi. This case highlights the diagnostic limitations in detecting P. cynomolgi, which shares high genetic similarity with P. vivax, leading to potential cross-reactivity and diagnostic inaccuracies. As P. cynomolgi emerges as the second zoonotic malaria species after P. knowlesi capable of infecting humans in Southeast Asia, improved diagnostic methods are urgently needed. Enhanced molecular diagnostics and comprehensive epidemiological studies are essential to elucidate transmission dynamics, assess public health implications, and inform effective malaria control strategies.

Macaques, Macaca fascicularis , are a known reservoir of Plasmodium knowlesi, the agent of simian malaria which is the predominant zoonotic species affecting humans in Malaysia and other Southeast Asian countries. Recently, a naturally acquired human infection of another simian malaria parasite, P. cynomolgi has been reported. Thus, it is crucial to study the distribution of simian Plasmodium infections with particular attention to the macaques. Four hundred and nineteen (419) long-tailed macaques ( Macaca fascicularis ) were trapped in selected areas where human cases of P. knowlesi and P. cynomolgi have been reported. Nested polymerase chain reaction (PCR) was conducted to identify the Plasmodium spp., and circumsporozoite protein (CSP) genes of P. knowlesi samples were sequenced. Plasmodium cynomolgi infection was shown to be the most prevalent among the macaque population (68.4%). Although 50.6% of analyzed samples contained single infections either with P. knowlesi , P. cynomolgi , P. inui , P. coatneyi , or P. fieldi , mixed infections with double, triple, quadruple, and all 5 species were also detected. Infection with P. cynomolgi and P. knowlesi were the highest among Malaysian macaques in areas where humans and macaques are in close contact. The risk of zoonotic infection in these areas needs to be addressed since the number of zoonotic malaria cases is on the rise. With the elimination of human malaria, the risk of humans being infected with simian malaria is very high and steps should be taken to mitigate this issue. Les macaques, Macaca fascicularis , sont un réservoir connu de Plasmodium knowlesi , l’agent du paludisme simien qui est l’espèce zoonotique prédominante affectant les humains en Malaisie et dans d’autres pays d’Asie du Sud-Est. Récemment, une infection humaine acquise naturellement par un autre parasite du paludisme simien, P. cynomolgi , a été signalée. Ainsi, il est crucial d’étudier la distribution des infections simiennes à Plasmodium avec une attention particulière pour les macaques. Quatre cent dix-neuf (419) macaques à longue queue ( Macaca fascicularis ) ont été piégés dans des zones sélectionnées où des cas humains de P. knowlesi et P. cynomolgi avaient été signalés. La réaction en chaîne par polymérase (PCR) nichée a été menée pour identifier les Plasmodium spp. et les gènes de la protéine circumsporozoïte (CSP) des échantillons de P. knowlesi ont été séquencés. L’infection à P. cynomolgi s’est avérée la plus répandue parmi la population de macaques (68,4 %). Bien que 50,6 % des échantillons analysés montraient des infections simples avec soit P. knowlesi , P. cynomolgi , P. inui , P. coatneyi ou P. fieldi , des infections mixtes avec deux, trois, quatre ou même les cinq espèces ont également été détectées. L’infection par P. cynomolgi et P. knowlesi était la plus élevée parmi les macaques malais dans les zones où les humains et les macaques sont en contact étroit. Le risque d’infection zoonotique dans ces zones doit être pris en compte car le nombre de cas de paludisme zoonotique est en augmentation. Avec l’élimination du paludisme humain, le risque d’être infecté par le paludisme simien est très élevé et des mesures doivent être prises pour atténuer ce problème.

Please note that an author has been erroneously omitted from the author list of the published article [1].Please note that an author has been erroneously omitted from the author list of the published article [1].