Plasmodium Cynomolgi Research Articles

Plasmodium cynomolgi: What Should We Know?

Even though malaria has markedly reduced its global burden, it remains a serious threat to people living in or visiting malaria-endemic areas. The six Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri and Plasmodium knowlesi) are known to associate with human malaria by the Anopheles mosquito. Highlighting the dynamic nature of malaria transmission, the simian malaria parasite Plasmodium cynomolgi has recently been transferred to humans. The first human natural infection case of P. cynomolgi was confirmed in 2011, and the number of cases is gradually increasing. It is assumed that it was probably misdiagnosed as P. vivax in the past due to its similar morphological features and genome sequences. Comprehensive perspectives that encompass the relationships within the natural environment, including parasites, vectors, humans, and reservoir hosts (macaques), are required to understand this zoonotic malaria and prevent potential unknown risks to human health.

Loop-Mediated Isothermal Amplification for Diagnosis of Zoonotic Malaria.

Zoonotic malaria, caused by Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium coatneyi, and Plasmodium inui, is a significant global health concern. The gold standard microscopy, while widely used for malaria diagnosis, faces limitations in differentiating between malaria species. Polymerase chain reaction (PCR), despite its accuracy, is characterized by high costs and time-consuming procedures. This study aims to develop and validate a rapid and accurate diagnostic test for detecting four simian Plasmodium species by using loop-mediated isothermal amplification (LAMP). Loop-mediated isothermal amplification is a cost-effective and faster molecular testing alternative for malaria diagnosis. The project involved designing specific primers, testing sensitivity and specificity against various parasites (including human Plasmodium species, protozoa, and helminths), and evaluating the LAMP assay using 60 macaque samples infected with simian Plasmodium. The LAMP assay exhibited a sensitivity profile enabling the detection of P. knowlesi, P. coatneyi, and P. cynomolgi across a concentration gradient from 5 × 108 down to 5 × 105 parasites/µL. Notably, P. inui was detectable at 5 × 108 parasites/µL. Furthermore, the specificity of the primer tailored for the four simian Plasmodium species was proven, as it produced a positive amplification exclusively for the respective target species and generated negative results for nontarget species. The results indicated that the LAMP assay is capable of detecting simian Plasmodium within a short span of 60 minutes, without any false positives from other samples. This new test has the potential to revolutionize malaria diagnosis, surveillance, and control, thereby mitigating the impact of zoonotic malaria in regions of endemicity.

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

BackgroundNatural 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. MethodsBy 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. ResultsThere 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. ConclusionThis 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.

Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs.

The development of novel antiplasmodial compounds with broad-spectrum activity against different stages of Plasmodium parasites is crucial to prevent malaria disease and parasite transmission. This study evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds inhibited blood stage proliferation of drug-resistant or sensitive strains of Plasmodium falciparum in the low micromolar to nanomolar range. Interestingly, CB-41 exhibited prophylactic activity against hypnozoites and liver schizonts in Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Isobologram assays demonstrated synergistic interactions between CB-61 and the FDA-approved antimalarial drugs, clindamycin and halofantrine. Testing of six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target and no cytotoxicity in HepG2 liver cells. CB compounds are promising candidates for further development as antimalarial drugs against multidrug-resistant parasites, which could also prevent malaria transmission.

Superior protection in a relapsing Plasmodium cynomolgi rhesus macaque model by a chemoprophylaxis with sporozoite immunization regimen with atovaquone-proguanil followed by primaquine

BackgroundTo gain a deeper understanding of protective immunity against relapsing malaria, this study examined sporozoite-specific T cell responses induced by a chemoprophylaxis with sporozoite (CPS) immunization in a relapsing Plasmodium cynomolgi rhesus macaque model.MethodsThe animals received three CPS immunizations with P. cynomolgi sporozoites, administered by mosquito bite, while under two anti-malarial drug regimens. Group 1 (n = 6) received artesunate/chloroquine (AS/CQ) followed by a radical cure with CQ plus primaquine (PQ). Group 2 (n = 6) received atovaquone-proguanil (AP) followed by PQ. After the final immunization, the animals were challenged with intravenous injection of 104P. cynomolgi sporozoites, the dose that induced reliable infection and relapse rate. These animals, along with control animals (n = 6), were monitored for primary infection and subsequent relapses. Immunogenicity blood draws were done after each of the three CPS session, before and after the challenge, with liver, spleen and bone marrow sampling and analysis done after the challenge.ResultsGroup 2 animals demonstrated superior protection, with two achieving protection and two experiencing partial protection, while only one animal in group 1 had partial protection. These animals displayed high sporozoite-specific IFN-γ T cell responses in the liver, spleen, and bone marrow after the challenge with one protected animal having the highest frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. Partially protected animals also demonstrated a relatively high frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. It is important to highlight that the second animal in group 2, which experienced protection, exhibited deficient sporozoite-specific T cell responses in the liver while displaying average to high T cell responses in the spleen and bone marrow.ConclusionsThis research supports the notion that local liver T cell immunity plays a crucial role in defending against liver-stage infection. Nevertheless, there is an instance where protection occurs independently of T cell responses in the liver, suggesting the involvement of the liver's innate immunity. The relapsing P. cynomolgi rhesus macaque model holds promise for informing the development of vaccines against relapsing P. vivax.

The Plasmodium vivax MSP1P-19 is involved in binding of reticulocytes through interactions with the membrane proteins band3 and CD71

The parasite Plasmodium vivax preferentially invades human reticulocytes. Its merozoite surface protein 1 paralog (PvMSP1P), particularly the 19-kDa C-terminal region (PvMSP1P-19), has been shown to bind to reticulocytes and this binding can be inhibited by antisera obtained by PvMSP1P-19 immunization. The molecular mechanism of interactions between PvMSP1P-19 and reticulocytes during P. vivax invasion, however, remains unclear. In this study, we analyzed the ability of MSP1P-19 to bind to different concentrations of reticulocytes and confirmed its reticulocyte preference. LC-MS analysis was used to identify two potential reticulocyte receptors, band3 and CD71, that interact with MSP1P-19. Both PvMSP1P-19 and its sister taxon Plasmodium cynomolgi MSP1P-19 (PcMSP1P-19) were found to bind to the extracellular loop (loop 5) of band3, where the interaction of MSP1P-19 with band3 was chymotrypsin-sensitive. Antibodies against band3-P5, CD71, and MSP1P-19 reduced the binding activity of PvMSP1P-19 and PcMSP1P-19 to reticulocytes, while MSP1P-19 proteins inhibited P. falciparum invasion in vitro in a concentration-dependent manner. To sum up, identification and characterization of the reticulocyte receptor is important for understanding the binding of reticulocytes by MSP1P-19.

Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

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.

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.

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites

BackgroundThe infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria.ResultsThe liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples’ features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria.ConclusionsA CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.Graphical

Overview of Plasmodium spp. and Animal Models in Malaria Research.

Malaria is a parasitic disease caused by protozoan species of the genus Plasmodium and transmitted by female mosquitos of the genus Anopheles and other Culicidae. Most of the parasites of the genus Plasmodium are highly species specific with more than 200 species described affecting different species of mammals, birds, and reptiles. Plasmodium species strictly affecting humans are P. falciparum, P. vivax, P. ovale, and P. malariae. More recently, P. knowlesi and other nonhuman primate plasmodia were found to naturally infect humans. Currently, malaria occurs mostly in poor tropical and subtropical areas of the world, and in many of these countries it is the leading cause of illness and death. For more than 100 y, animal models, have played a major role in our understanding of malaria biology. Avian Plasmodium species were the first to be used as models to study human malaria. Malaria parasite biology and immunity were first studied using mainly P. gallinaceum and P. relictum. Rodent malarias, particularly P. berghei and P. yoelii, have been used extensively as models to study malaria in mammals. Several species of Plasmodium from nonhuman primates have been used as surrogate models to study human malaria immunology, pathogenesis, candidate vaccines, and treatments. Plasmodium cynomolgi, P. simiovale, and P. fieldi are important models for studying malaria produced by P. vivax and P. ovale, while P. coatneyi is used as a model for study- ing severe malaria. Other nonhuman primate malarias used in research are P. fragile, P. inui, P. knowlesi, P. simium, and P. brasilianum. Very few nonhuman primate species can develop an infection with human malarias. Macaques in general are resistant to infection with P. falciparum, P. vivax, P. malariae, and P. ovale. Only apes and a few species of New World monkeys can support infection with human malarias. Herein we review the most common, and some less common, avian, reptile, and mammal plasmodia species used as models to study human malaria.

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.

Transfection Models to Investigate Plasmodium vivax-Type Dormant Liver Stage Parasites.

Plasmodium vivax causes the second highest number of malaria morbidity and mortality cases in humans. Several biological traits of this parasite species, including the formation of dormant stages (hypnozoites) that persist inside the liver for prolonged periods of time, present an obstacle for intervention measures and create a barrier for the elimination of malaria. Research into the biology of hypnozoites requires efficient systems for parasite transmission, liver stage cultivation and genetic modification. However, P. vivax research is hampered by the lack of an in vitro blood stage culture system, rendering it reliant on in vivo-derived, mainly patient, material for transmission and liver stage culture. This has also resulted in limited capability for genetic modification, creating a bottleneck in investigations into the mechanisms underlying the persistence of the parasite inside the liver. This bottleneck can be overcome through optimal use of the closely related and experimentally more amenable nonhuman primate (NHP) parasite, Plasmodium cynomolgi, as a model system. In this review, we discuss the genetic modification tools and liver stage cultivation platforms available for studying P. vivax persistent stages and highlight how their combined use may advance our understanding of hypnozoite biology.

The potential for zoonotic malaria transmission in five areas of Indonesia inhabited by non-human primates

BackgroundIndonesia is home to many species of non-human primates (NHPs). Deforestation, which is still ongoing in Indonesia, has substantially reduced the habitat of NHPs in the republic. This has led to an intensification of interactions between NHPs and humans, which opens up the possibility of pathogen spillover. The aim of the present study was to determine the prevalence of malarial parasite infections in NHPs in five provinces of Indonesia in 2022. Species of the genus Anopheles that can potentially transmit malarial pathogens to humans were also investigated.MethodsAn epidemiological survey was conducted by capturing NHPs in traps installed in several localities in the five provinces, including in the surroundings of a wildlife sanctuary. Blood samples were drawn aseptically after the NHPs had been anesthetized; the animals were released after examination. Blood smears were prepared on glass slides, and dried blood spot tests on filter paper. Infections with Plasmodium spp. were determined morphologically from the blood smears, which were stained with Giemsa solution, and molecularly through polymerase chain reaction and DNA sequencing using rplU oligonucleotides. The NHPs were identified to species level by using the mitochondrial cytochrome c oxidase subunit I gene and the internal transcribed spacer 2 gene as barcoding DNA markers. Mosquito surveillance included the collection of larvae from breeding sites and that of adults through the human landing catch (HLC) method together with light traps.ResultsAnalysis of the DNA extracted from the dried blood spot tests of the 110 captured NHPs revealed that 50% were positive for Plasmodium, namely Plasmodium cynomolgi, Plasmodium coatneyi, Plasmodium inui, Plasmodium knowlesi and Plasmodium sp. Prevalence determined by microscopic examination of the blood smears was 42%. Species of the primate genus Macaca and family Hylobatidae were identified by molecular analysis. The most common mosquito breeding sites were ditches, puddles and natural ponds. Some of the Anopheles letifer captured through HLC carried sporozoites of malaria parasites that can cause the disease in primates.ConclusionsThe prevalence of malaria in the NHPs was high. Anopheles letifer, a potential vector of zoonotic malaria, was identified following its collection in Central Kalimantan by the HLC method. In sum, the potential for the transmission of zoonotic malaria in several regions of Indonesia is immense.Graphical

Combining isothermal recombinase polymerase amplification with lateral flow assay for diagnosis of P. cynomolgi malaria infection.

Plasmodium cynomolgi is a nonhuman primate parasite that causes malaria in humans and is transmitted by the Anopheles mosquito. Macaques, the natural hosts of P. cynomolgi, are widely distributed in Asia, especially in Southeast Asia. Anthropogenic land-use changes and wildlife habitat reduction due to local environmental changes, deforestation, urban expansion, and construction increased the frequency of human-macaque-vector interactions and facilitated the emergence of zoonotic malaria, causing an exponential increase in the infection rates in this area. Although microscopic tools are the gold standard for malaria diagnosis, they have very low sensitivity. Therefore, disease control and prevention require rapid, sensitive and accurate diagnostic tests. This study aims to develop a diagnostic method using a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method to specifically diagnose P. cynomolgi. Laboratory validation determined the method's sensitivity and specificity compared to the nested PCR method. The lower limit of detection was 22.14 copies/μl of recombinant plasmid per reaction. The combination method represented 81.82% sensitivity and 94.74% specificity compared to the nested PCR. The diagnostic testing developed in this study combines a recombinase polymerase amplification (RPA) and a lateral flow (LF) strip, offering rapid high sensitivity and specificity. Further development of this technique could make it a promising method for detecting P. cynomolgi.

Epigenetically regulated RNA-binding proteins signify malaria hypnozoite dormancy

Dormancy enables relapsing malaria parasites, such as Plasmodium vivax and cynomolgi, to survive unfavorable conditions. It is enabled by hypnozoites, parasites remaining quiescent inside hepatocytes before reactivating and establishing blood-stage infection. We integrate omics approaches to explore gene-regulatory mechanisms underlying hypnozoite dormancy. Genome-wide profiling of activating and repressing histone marks identifies a few genes that get silenced by heterochromatin during hepatic infection of relapsing parasites. By combining single-cell transcriptomics, chromatin accessibility profiling, and fluorescent in situ RNA hybridization, we show that these genes are expressed in hypnozoites and that their silencing precedes parasite development. Intriguingly, these hypnozoite-specific genes mainly encode proteins with RNA-binding domains. We hence hypothesize that these likely repressive RNA-binding proteins keep hypnozoites in a developmentally competent but dormant state and that heterochromatin-mediated silencing of the corresponding genes aids reactivation. Exploring the regulation and exact function of these proteins hence could provide clues for targeted reactivation and killing of these latent pathogens.

High transmission efficiency of the simian malaria vectors and population expansion of their parasites Plasmodium cynomolgi and Plasmodium inui.

The elimination of malaria in Southeast Asia has become more challenging as a result of rising knowlesi malaria cases. In addition, naturally occurring human infections with other zoonotic simian malaria caused by Plasmodium cynomolgi and Plasmodium inui adds another level of complexity in malaria elimination in this region. Unfortunately, data on vectors which are responsible for transmitting this zoonotic disease is very limited. We conducted longitudinal studies to investigate the entomological parameters of the simian malaria vectors and to examine the genetic diversity and evolutionary pattern of their simian Plasmodium. All the captured Anopheles mosquitoes were dissected to examine for the presence of oocysts, sporozoites and to determine the parous rate. Our study revealed that the Anopheles Leucosphyrus Group mosquitoes are highly potential competent vectors, as evidenced by their high rate of parity, survival and sporozoite infections in these mosquitoes. Thus, these mosquitoes represent a risk of human infection with zoonotic simian malaria in this region. Haplotype analysis on P. cynomolgi and P. inui, found in high prevalence in the Anopheles mosquitoes from this study, had shown close relationship between simian Plasmodium from the Anopheles mosquitoes with its vertebrate hosts. This directly signifies the ongoing transmission between the vector, macaques, and humans. Furthermore, population genetic analysis showed significant negative values which suggest that both Plasmodium species are undergoing population expansion. With constant microevolutionary processes, there are potential for both P. inui and P. cynomolgi to emerge and spread as a major public health problem, following the similar trend of P. knowlesi. Therefore, concerted vector studies in other parts of Southeast Asia are warranted to better comprehend the transmission dynamics of this zoonotic simian malaria which eventually would aid in the implementation of effective control measures in a rapidly changing environment.

An active and targeted survey reveals asymptomatic malaria infections among high-risk populations in Mondulkiri, Cambodia

BackgroundMalaria is a mosquito-borne disease that is one of the most serious public health issues globally and a leading cause of mortality in many developing countries worldwide. Knowing the prevalence of both symptomatic and asymptomatic malaria on a subnational scale allows for the estimation of the burden of parasitaemia present in the transmission system, enabling targeting and tailoring of resources towards greater impact and better use of available capacity. This study aimed to determine the PCR-based point prevalence of malaria infection, by parasite species, among three high-risk populations in Mondulkiri province, Cambodia: forest rangers, forest dwellers, and forest goers.MethodsA cross-sectional survey was performed during the transmission season in November and December 2021. Blood samples collected on filter paper from participants (n = 1301) from all target groups were screened for Plasmodium spp using PCR.ResultsMalaria prevalence among all study participants was 6.7% for any Plasmodium species. Malaria prevalence in the forest ranger group was 8.1%, was 6.8% in forest goers, and 6.4% in forest dwellers; all infections were asymptomatic. Plasmodium vivax was detected in all participant groups, while the few Plasmodium falciparum infections were found in goers and dwellers. 81% of all infections were due to P. vivax, 9% were due to P. falciparum, 3% due to Plasmodium cynomolgi, and the rest (7%) remained undefined. Gender was associated with malaria infection prevalence, with male participants having higher odds of malaria infection than female participants (OR = 1.69, 95% CI 1.08–2.64). Passively collected malaria incidence data from the Cambodian government were also investigated. Health facility-reported malaria cases, based on rapid diagnostic tests, for the period Jan-Dec 2021 were 521 Plasmodium vivax (0.89% prevalence), 34 P. falciparum (0.06%) and four P. falciparum + mixed (0.01%)—a total of 559 cases (0.95%) for all of Mondulkiri.ConclusionThis reservoir of asymptomatic parasitaemia may be perpetuating low levels of transmission, and thus, new strategies are required to realize the goal of eliminating malaria in Cambodia by 2025.

Lateral Flow Recombinase Polymerase Amplification Assays for the Detection of Human Plasmodium Species.

This study highlights the development of two lateral flow recombinase polymerase amplification assays for the diagnosis of human malaria. The lateral flow cassettes contained test lines that captured biotin-, 6-carboxyfluorescein, digoxigenin-, cyanine 5-, and dinitrophenyl-labeled amplicons. The overall process can be completed in 30 minutes. Recombinase polymerase amplification coupled with lateral flow had a detection limit of 1 copy/µL for Plasmodium knowlesi, Plasmodium vivax, and Plasmodium falciparum. No cross-reactivity was observed among nonhuman malaria parasites such as Plasmodium coatneyi, Plasmodium cynomolgi, Plasmodium brasilanium, Plasmodium inui, Plasmodium fragile, Toxoplasma gondii, Sarcocystis spp., Brugia spp., and 20 healthy donors. It is rapid, highly sensitive, robust, and easy to use. The result can be read without the need for special equipment and thus has the potential to serve as an effective alternative to polymerase chain reaction methods for the diagnosis of malaria.

A novel multiplex qPCR assay for clinical diagnosis of non-human malaria parasites-Plasmodium knowlesi and Plasmodium cynomolgi.

The imminent risk of zoonoses of non-human malaria parasites is not far from reality in India, as has been observed in the case of Plasmodium knowlesi (Pk), and so is possible with P. cynomolgi (Pc), already reported from South East Asian countries. Therefore, a novel multiplex qPCR assay was developed and evaluated for detection of non-human malaria parasites- Pk and Pc in populations at risk. The qPCR primers were designed in-house with fluorescence labeled probes (HEX for Pk and FAM for Pc). DNA samples of Pk and Pc were used as templates and further the qPCR assay was evaluated in 250 symptomatic and asymptomatic suspected human blood samples from malaria endemic areas of North Eastern states of India. The qPCR assay successfully amplified the target 18S rRNA gene segment from Pk and Pc and was highly specific for Pk and Pc parasites only, as no cross reactivity was observed with P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale (Po). Standard curves were generated to estimate the limit of detection (LOD) of Pk and Pc parasites DNA (0.00275 & 0.075 ng/μl, respectively). Due to COVID-19 pandemic situation during 2020-21, the sample accessibility was difficult, however, we managed to collect 250 samples. The samples were tested for Pf and Pv using conventional PCR- 14 Pf and 11 Pv infections were observed, but no Pk and Pc infections were detected. For Pk infections, previously reported conventional PCR was also performed, but no Pk infection was detected. The multiplex qPCR assay was observed to be robust, quick, cost-effective and highly sensitive as compared to the currently available conventional PCR methods. Further validation of the multiplex qPCR assay in field setting is desirable, especially from the high-risk populations. We anticipate that the multiplex qPCR assay would prove to be a useful tool in mass screening and surveillance programs for detection of non-human malaria parasites toward the control and elimination of malaria from India by 2030.