Acanthamoeba keratitis is a serious infection with blinding consequences and often associated with contact lens wear. Early diagnosis, followed by aggressive topical application of drugs, is a prerequisite in successful treatment, but even then prognosis remains poor. Several drugs have shown promise, including chlorhexidine gluconate; however, host cell toxicity at physiologically relevant concentrations remains a challenge. Nanoparticles, subcolloidal structures ranging in size from 10 to 100 nm, are effective drug carriers for enhancing drug potency. The overall aim of the present study was to determine whether conjugation with gold nanoparticles enhances the antiacanthamoebic potential of chlorhexidine. Gold-conjugated chlorhexidine nanoparticles were synthesized. Briefly, gold solution was mixed with chlorhexidine and reduced by adding sodium borohydride, resulting in an intense deep red color, indicative of colloidal gold-conjugated chlorhexidine nanoparticles. The synthesis was confirmed using UV-visible spectrophotometry that shows a plasmon resonance peak of 500 to 550 nm, indicative of gold nanoparticles. Further characterization using matrix-assisted laser desorption ionization-mass spectrometry showed a gold-conjugated chlorhexidine complex at m/z 699 ranging in size from 20 to 100 nm, as determined using atomic force microscopy. To determine the amoebicidal and amoebistatic effects, amoebae were incubated with gold-conjugated chlorhexidine nanoparticles. For controls, amoebae also were incubated with gold and silver nanoparticles alone, chlorhexidine alone, neomycin-conjugated nanoparticles, and neomycin alone. The findings showed that gold-conjugated chlorhexidine nanoparticles exhibited significant amoebicidal and amoebistatic effects at 5 μM. Amoebicidal effects were observed by parasite viability testing using a Trypan blue exclusion assay and flow-cytometric analysis using propidium iodide, while amoebistatic effects were observed using growth assays. In contrast, chlorhexidine alone, at a similar concentration, showed limited effects. Notably, neomycin alone or conjugated with nanoparticles did not show amoebicidal or amoebistatic effects. Pretreatment of A. castellanii with gold-conjugated chlorhexidine nanoparticles reduced amoeba-mediated host cell cytotoxicity from 90% to 40% at 5 μM. In contrast, chlorhexidine alone, at similar concentrations, had no protective effects for the host cells. Similarly, amoebae treated with neomycin alone or neomycin-conjugated nanoparticles showed no protective effects. Overall, these findings suggest that gold-conjugated chlorhexidine nanoparticles hold promise in the improved treatment of A. castellanii keratitis.
Megaselia scalaris (Loew) is a cosmopolitan polyphagous small fly with the ability of exploiting variety of ecological niches. Different life history stages act as detritivore, parasite, and parasitoid of wider spectrum of plant and animal matter under natural and laboratory conditions. Here, for the first time we present the opportunistic parasitism of M. scalaris on Otobius megnini, which act as a vector of Q fever and is capable of causing paralysis, toxic conditions, otoacariasis and otitis in humans and other animals. Tick samples from the ear canals of 14 thoroughbred horses were brought to the laboratory and several days later, larvae of M. scalaris were found feeding on immature stages of O. megnini. When the development was completed pupae were found attached to adult ticks and all nymphs were found dead. This context reveals the capability of M. scalaris surviving on O. megnini and the risk of their invading ear canals of horses.
Studies on parasite populations in Antarctic soils are scarce and thus little is known about the threat of these parasites towards either the natural fauna or human visitors. However, human presence in Antarctica, mainly through research and tourism, keeps increasing over time, potentially exposing visitors to zoonotic infections from Antarctic wildlife and environment. Most available literature to date has focused on faecal samples from Antarctic vertebrates. Therefore, this study addressed the possible presence of parasites in Antarctic soil that may be infectious to humans. Soil samples were obtained from five locations on Signy Island (South Orkney Islands, maritime Antarctic), namely North Point and Gourlay Peninsula (penguin rookeries), Pumphouse (relic coal-powered pump house), Jane Col (barren high altitude fellfield) and Berntsen Point (low altitude vegetated fellfield close to current research station). Approximately 10% of the soil samples (14/135) from 3 out of the 5 study sites had parasites which included Diphyllobotridae spp. eggs, Cryptosporidium sp., an apicomplexan protozoa (gregarine), Toxoplasma gondii, helminths (a cestode, Tetrabothrius sp., and a nematode larva) and mites. The presence of parasites in the 3 sites are most likely due to the presence of animal and human activities as two of these sites are penguin rookeries (North Point and Gourlay Peninsula) while the third site (Pumphouse Lake) has human activity. While some of the parasite species found in the soil samples appear to be distinctive, there were also parasites such as Cryptosporidium and Toxoplasma gondii that have a global distribution and are potentially pathogenic.
Malaria is one of the most dangerous infectious diseases due to its high infection and mortality rates, especially in the tropical belt. Plasmodium falciparum (P. falciparum), the most virulent malaria parasite in humans, was recently reported to develop resistance against the final efficient antimalarial drug, artemisinin. Little is known about the resistance mechanisms, which further complicates the problem as a proper counteraction is unable to be taken. Hence, the understanding of drug mode of action and its molecular target is valuable knowledge that needs to be considered to develop the next generation of antimalarial drugs. P. falciparum protein kinase (Pf PK) is an attractive target for antimalarial chemotherapy due to its vital roles in all P. falciparum life stages. Moreover, overall structural differences and the presence of unique Pf PKs that are absent in human kinome, suggesting specific inhibition of Pf PK without affecting human cells is achievable. To date, at least 86 eukaryotic protein kinases have been identified in P. falciparum kinome, by which less than 40 were validated as potential targets at the erythrocytes stage. In this review, recent progress of the furthest validated Pf PKs; Pf Nek-1, Pf CDPK1, Pf CDPK4, Pf PKG, and Pf CLK-3 will be briefly discussed.
Chloroquine resistance transporter of Plasmodium falciparum (PfCRT) is a food vacuolar transmembrane protein that mediates susceptibility of the parasite to chloroquine. A mutation at K76T of the Pfcrt gene is a key determinant for chloroquine resistance phenotype. In the absence of drug pressure, in vitro growth rate of chloroquine-resistance parasites was outcompeted by wild-type parasites unless intragenic compensatory mutations occurred. Chloroquine-resistant P. falciparum bearing the Cam734 haplotype known to circulate in endemic areas of Cambodia bordering Thailand contains 9 mutations in Pfcrt and exhibits both chloroquine resistance and comparable growth rate to the chloroquine-sensitive 3D7 strain. To analyze the evolution of the Cam734 haplotype, codon-based analysis was performed by using the mixed effects model of evolution (MEME), branch-site random effects likelihood (BR-REL) and other related methods. Results revealed that the Cam734 haplotype has evolved distinctively from other known mutant haplotypes including the most common Dd2 haplotype in Southeast Asia. Evidence of episodic positive selection was detected at codon 144, characterized by c.[430G>T; 431C>T] (p.A144F), known to be indispensable for both chloroquine resistance and restoration of growth rate of the parasites. To survey the prevalence of mutations at codons 76 and 144 in Pfcrt among Thai isolates, restriction fragment analysis of 548 P. falciparum isolates collected from six endemic provinces of Thailand during 1991 and 2016 was performed. The 144F Pfcrt mutant was detected in 7 (1.28%) isolates. All Thai isolates analyzed herein harbored a mutation at codon 76 whilst the wild-type parasite was not found. The low prevalence of isolates bearing the mutation 144F in PfCRT could imply little or lack of survival advantage of this mutant in endemic areas of Thailand where the wild-type parasites seem to be absent or extremely rare.
Strongyloidiasis is a mysterious yet important parasitic disease that is hard to diagnose. While microscopic examination remains a "controversial" gold standard method, improved diagnosis is achieved through confirmatory assays with serological and/or molecular diagnostic approaches. In the current serodiagnosis of strongyloidiasis, recombinant proteins have been adopted in place of the use of native parasite antigens, although the availability of diagnostically potential proteins are still limited. Here, we introduce a novel Strongyloides recombinant protein that is uniquely attached to two different short peptide tags as a potential diagnostic biomarker for serodiagnosis of strongyloidiasis, namely lysine (7K) and aspartic acid (7D). The work presented focus on improving the yield and purity of the previously unexpressed recombinant protein. Preliminary diagnostic evaluation of the recombinant favors Ss3a7K protein owing to its higher antigenicity performance with 80% sensitivity and 100% specificity, respectively.
Introduction. Despite the efforts of the malaria control programme, malaria morbidity is still a common health problem in Yemen, with 60% of the population at risk. Plasmodium falciparum is responsible for 99% of malaria cases. The emergence in Yemen of parasite resistance to chloroquine (CQ) prompted the adoption of artemisinin combination therapy (ACT) in 2009, which involves the use of artesunate plus sulphadoxine-pyrimethamine (AS + SP). However, CQ was retained as the drug of choice for vivax malaria. To assess the impact of the change in the malaria treatment policy five years after its introduction, the present study investigated the mutations in the CQ resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes. Method. A molecular investigation of 10 codons of pfcrt (72-76, 220, 271, 326, 356, and 371) and five codons of pfmdr1 (86, 184, 1034, 1042, and 1246) was conducted on P. falciparum isolates from districts with the highest malaria endemicity in the Hodeidah and Al-Mahwit governorates in Tehama region, Yemen. A total of 86 positive cases of falciparum monoinfection were investigated for the presence of mutations related to CQ and other antimalarials using a PCR-RFLP assay. Results. There was a wide prevalence of pfcrt gene mutations with the pfcrt 76T CQ resistance marker being predominant (97.7%). The prevalence of other pfcrt mutations varied from high (75E: 88%) to moderate (74I: 79.1%, 220S: 69.8%, 271E and 371I: 53.5%) or low (326S: 36%, 72S: 10.5%). Mutated pfcrt 72-76 amino acids haplotypes were highly prevalent (98.8%). Among these, the CVIET classic, old-world African/Southeast Asian haplotype was the most predominant, and was mostly found in the isolates from the Khamis Bani Saad district of Al-Mahwit (93.1%) and the AdDahi district of Hodeidah (88.9%). However, it was only found in 26.3% of the isolates from the Bajil district of Hodeidah. Surprisingly, the SVMNT new-world South American haplotype was exclusively detected in 9.3% of the isolates from the Bajil district of Hodeidah. Mutations at Y184F of pfmdr1 were found in all isolates (100%) from all districts. The mutation for codons 1034C and 86Y were found only in the isolates from the AdDahi and Khamis Bani Saad districts. Overall, the AdDahi and Khamis Bani Saad districts were similar in terms of carrying most of the mutations in the pfcrt and pfmdr1 genes, while there was a lower prevalence of mutation in the isolates from the Bajil district. Conclusion. The high prevalence of mutations in pfcrt 5 years after the official cessation of CQ use against P. falciparum suggests that there is sustained CQ pressure on P. falciparum isolates in the study area. Moreover, the low prevalence of mutations in the pfmdr1 gene could be a good indicator of the high susceptibility of P. falciparum isolates to antimalarials other than CQ. A new strategy to ensure the complete nationwide withdrawal of CQ from the private drug market is recommended.
Anopheles mosquitoes transmit malaria which is one of the world's most threatening diseases. Anopheles dirus (sensu stricto) is among the main vectors of malaria in South East Asia. The mosquito innate immune response is the first line of defence against malaria parasites during its development. The immune deficiency (IMD) pathway, a conserved immune signaling pathway, influences anti-Plasmodium falciparum activity in Anopheles gambiae, An. stephensi and An. albimanus. The aim of the study was to determine the role of Rel2, an IMD pathway-controlled NF-kappaβ transcription factor, in An. dirus.
Mosquitoes are arthropods of huge medical and veterinary relevance, since they vector pathogens and parasites of public health importance, including malaria, dengue and Zika virus. Currently, nanotechnology is considered a potential eco-friendly approach in mosquito control research. We proposed a novel method of biofabrication of silver nanoparticles (AgNP) using chitosan (Ch) from crab shells. Ch-AgNP nanocomposite was characterized by UV-vis spectroscopy, FTIR, SEM, EDX and XRD. Ch-AgNP were tested against larvae and pupae of the malaria vector Anopheles stephensi obtaining LC50 ranging from 3.18 ppm (I) to 6.54 ppm (pupae). The antibacterial properties of Ch-AgNP were proved against Bacillus subtilis, Klebsiella pneumoniae and Salmonella typhi, while no growth inhibition was reported in assays conducted on Proteus vulgaris. Concerning non-target effects, in standard laboratory considtions the predation efficiency of Danio rerio zebrafishes was 68.8% and 61.6% against I and II instar larvae of A. stephensi, respectively. In a Ch-AgNP-contaminated environment, fish predation was boosted to 89.5% and 77.3%, respectively. Quantitative analysis of antioxidant enzymes SOD, CAT and LPO from hepatopancreas of fresh water crabs Paratelphusa hydrodromous exposed for 16 days to a Ch-AgNP-contaminated aquatic environment were conducted. Notably, deleterious effects of Ch-AgNP contaminating aquatic enviroment on the non-target crab P. hydrodromous were observed, particularly when doses higher than 8-10ppm are tested. Overall, this research highlights the potential of Ch-AGNP for the development of newer control tools against young instar populations of malaria mosquitoes, also highlighting some risks concerned the employ of nanoparticles in aquatic environments.
To assess stress level induced by multiple stressors in aquatic organism, biomarkers have been adopted as early warning indicator due to their high accuracy, rapidity, and sensitivity. We investigated the effects of ectoparasitic isopod infection on heavy metal bioaccumulation (Fe, Cu, Zn, and Cd) in the fish Nemipterus furcosus and profiled the expression of metallothionein (MT) and heat shock proteins 70 (HSP70) genes of the fish host. Sixty individuals (parasitized and nonparasitized with Cymothoa truncata) were collected from three sites differing in the levels of anthropogenic activities off the South China Sea. Our results revealed no significant difference in heavy metal concentrations between infected and nonparasitized fish. We observed a positive correlation between heavy metal bioaccumulation in the fish host and anthropogenic activities. Accordingly, expression analysis of MT genes in fish liver showed significant differences in expression level between sampling sites, with lowest level in the least exploited site (Batu Rakit). A reverse pattern in HSP70 gene expression was demonstrated in fish muscle, showing the highest expression at Batu Rakit. While cymothoid infection in N. furcosus had no significant impact on fish MT gene expression, it resulted in a reduction of HSP70 level in liver of parasitized fish. These findings highlight the putative roles of MT in heavy metal assessment. Future studies should determine the kinetics of cymothoid infection and other potential stressors in characterizing the HSP70 gene expression profile.
Cryptosporidium is an intestinal protozoan parasite which causes diarrhoea in animals and has recently been reported to cause similar symptoms in man. Cryptosporidiosis is a zoonotic infection and the first human case was reported in 1976.2 Since then the number of cases has increased by the hundreds especially among patients with acquired immuno-deficiency syndrome (AIDS), as a result of the severe symptoms that they cause in the im-munosuppressed patients and also due to the better screening techniques which have been developed resulting in the detection of cases.3 To date 20 species of Cryptosporidium have been reported but these cannot be differentiated morphologically. The differences are based mainly on the host from which the parasites were recovered. The species responsible for causing infection in man is Cryptosporidium parvum.4 Of the 20 species reported several are invalid because the oocysts of some of them were found to be the same as the sporocyst of Sarcocystis. It is now concluded that there are only two species that infect mammals (C. Muris and C. parvum). (Copied from article).
A four-month cross-sectional study found five species of parasitoids parasitizing puparia of filth flies breeding at the Taman Beringin landfill in Kepong and a poultry farm in Sungai Pelek, Sepang, Selangor. Effect of monthly rainfalls towards density of flies and percentage of parasitoids emerging from collected puparia were also analyzed. Spalangia sp. was the most common, consisting of Spalangia endius Walker, S. cameroni Perkins and S. gemina Boucek. Other parasitoids collected were Pachycrepoideus vindemmiae Rondani and Exoristobia phillipinensis Ashmead. The parasitized fly hosts were Musca domestica Linn. and Chrysomya megacephala Fabricius. S. endius was the most common parasitoid attacking M. domestica at both locations. M. domestica was the most common fly found at the Sg. Pelek poultry farm whereas C. megacephala was the most numerous at the Taman Beringin landfill. During heavy rainfall month of November 2003, density of flies were high whereas the emerging parasitoids were low at both landfill and poultry farm. The present study revealed the endemic presence of parasitoids especially S. endius in both poultry farm and garbage landfill and the potential of the parasitoid species in fly control in Malaysia.
Toxoplasmosis, caused by an intracellular protozoan parasite, Toxoplasma gondii, is widespread throughout the world. The disease is of major medical and veterinary importance, being a cause of congenital disease and abortion in humans and in domestic animals.[1] In addition, it has gained importance recently due to toxoplasma encephalitis in AIDS patients.[2] T. gondii was discovered 100 years ago. Its identification was rapidly followed by the recognition that it was a human pathogen. During the past 100 years, the spectrum of diseases caused by this ubiquitous pathogen has expanded to include both congenital and acute infections as well as the recognition of diseases caused by this pathogen in the immune-compromised host. Recent data on behavioural changes in animals due to chronic toxoplasmosis is leading to research on the effect of this pathogen on the behaviour of humans.[3] Experimental studies on T. gondii have resulted in it becoming a model organism for studies on host pathogen interactions. Integration of clinical and experimental data on T. gondii should continue to lead to important insights into improvements in diagnosis for clinical management and vaccine development for control of toxoplasmosis.
Rural health clinics in Sri Aman Division in Sarawak, Malaysia lack diagnostic tests for malaria. Many of the medical assistants in the clinic diagnose malaria solely on clinical ground. The study was to determine the sensitivity, specificity and positive predictive value of clinical diagnosis of rnahtria made by medical assistants using results of microscopy examination as gold standard. The study period was from September to December 2003. Three rural clinics without laboratory or diagnostic tests services and one urban clinic with laboratory facility serving malarious areas were selected. All patients clinically diagnosed as malaria by the attending medical assistants were included as clinical malaria cases. Blood slides were taken for examination of malaria parasites. Non clinical malaria cases were all other patients without clinical malaria for whom blood slides were taken for malaria parasites. Out of 362 patients included in the study, 75 were clinically diagnosed as having mahria. The sensitivity of headache, history of fever and chills/rigors to detect malaria cases was above 90% but the specificity and positive predictive value was low, below 40% and 20% respectively. The sensitivity, specificity and positive predictive value of malaria clinical diagnosis made by medical assistants was 82.1 %, 84 .4% and 30.7% respectively. Clinical diagnosis of malaria increases the slide positive rate by four folds. However the sensitivity, specincity and positive predictive value of clinical diagnosis by medical assistant and clinical characteristics was insufficient to enable each of them to be used alone to differentiate true malaria cases from non mahiria cases.
Malaria is a major public health problem in tropical and subtropical areas, caused by five
species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale andP. knowlesi) and is the leading cause of morbidity and mortality worldwide. We have developed molecular markers for three genes viz, Cytb, dhfr and Msp-1 gene and designed a protocol for rapid molecular diagnostics of the four malaria parasites prevalent in Southeast Asia. The new primers were used on the blood
samples containing Plasmodium parasites by conventional PCR. The result was compared with
the nested PCR of Singh et al. (2004) and the microscopy method. The result shows that the new
set of primers had successfully amplified all four human malaria parasite species. These primers
were 100% sensitive and more specific than microscopy and PCR identification using these
primers was faster than the nested PCR. These alternative primers should provide powerful and
rapid molecular diagnostic method for detecting Plasmodium species as well as providing reliable
data for epidemiology study. These primers have the potential to be combined and used in
multiplex PCR.
Screening of mud crab genus Scylla was conducted in four locations (Marudu Bay, Lundu, Taiping, Setiu) representing Malaysia. Scylla olivacea with abnormal primary and secondary sexual characters were prevalent (approximately 42.27% of the local screened S. olivacea population) in Marudu Bay, Sabah. A total of six different types of abnormalities were described. Crabs with type 1 and type 3 were immature males, type 2 and type 4 were mature males, type 5 were immature females and type 6 were mature females. The abdomen of all crabs with abnormalities were dented on both sides along the abdomen's middle line. Abnormal crabs showed significant variation in their size, weight, abdomen width and/or gonopod or pleopod length compared to normal individuals. The mean body weight of abnormal crabs (type 1-5) were higher than normal crabs with smaller body size, while females with type 6 abnormality were always heavier than the normal counterparts at any given size. Sacculinid's externa were observed in the abdomen of crabs with type 4 and type 6 abnormalities. The presence of embryos within the externa and subsequent molecular analysis of partial mitochondrial COI region confirmed the rhizocephalan parasite as Sacculina beauforti. Future in-depth descriptions of the life cycle and characteristics of S. beauforti are recommended as it involves a commercially important edible crab species and the effect on human health from the consumption of crabs is of crucial concern.
Within host communities, related species are more likely to share common parasitic agents, and as a result, morphological similarities have led researchers to conclude that parasites infecting closely related hosts within a community represent a single species. However, genetic diversity within parasite genera and host range remain poorly investigated in most systems. Strongyloides is a genus of soil-transmitted nematode that has been reported from several primate species in Africa and Asia, and has been estimated to infect hundreds of millions of people worldwide, although no precise estimates are available. Here we describe a case of infection with a cryptic species of Strongyloides in a Bornean (Philippine) slow loris (Nycticebus menagensis) living within a diverse community of several primate species in the Lower Kinabatangan Wildlife Sanctuary, Malaysian Borneo. Fresh fecal samples were collected from five primate species and nematode larvae cultured from these samples were selected for phylogenetic analyses. Sequences obtained for most larvae were identified as S. fuelleborni, grouping into three different clusters and showing no aggregation within specific hosts or geographic location. In contrast, a set of parasite sequences obtained from a slow loris clustered closely with S. stercoralis into a different group, being genetically distinct to sequences reported from other primate hosts, humans included. Our results suggest that although S. fuelleborni infects all haplorrhines sampled in this primate community, a different species might be infecting the slow loris, the only strepsirrhine in Borneo and one of the least studied primates in the region. Although more data are needed to support this conclusion, we propose that Strongyloides species in primates might be more diverse than previously thought, with potential implications for ecological and evolutionary host-parasite associations, as well as epidemiological dynamics.
The emergence of primate malaria known as Plasmodium knowlesi in humans, which is always misdiagnosed by microscopy as P. malariae, has contribute to the needs of nucleic acid based technology to be applied in detection and differentiation of malaria parasites. The target DNA sequence of the 18SrRNA gene was amplified by a nested PCR assay for detection and identification of Plasmodium species in 31 Giemsa-stained blood smears examined as P. malariae. The assay demonstrated three samples identified as positive to genus-specific primers but negative to all species-specific primers. Three cases of misdiagnosed species were detected. The samples were diagnosed as P. malariae microscopically, but detected as P. falciparum by PCR assay. Twenty five out of 31 samples were detected as P. knowlesi. None of the samples diagnosed microscopically as P. malariae were identified as P. malariae with the nested PCR assay. Over 80.6% of all malaria cases in this study showed naturally acquired P. knowlesi infections.
A morphological and molecular phylogenetic study of proteocephalid tapeworms of the genus Acanthotaenia von Linstow, 1903, parasites of monitors (Varanidae), was carried out. The type species, A. shipleyi von Linstow, 1903, which was originally described based on an immature specimen from Sri Lanka, is redescribed based on new material from the type host, Varanus salvator, in Sri Lanka, Malaysia, and Vietnam, and its neotype is designated. In addition, Acanthotaenia susanae n. sp. is described from Varanus nebulosus in Vietnam. The new species differs from congeners by the large size of the scolex, width of the rostellum and the number of testes. New molecular data (sequences of lsrDNA and cox1) revealed Acanthotaenia paraphyletic with the inclusion of Australotaenia bunthangi de Chambrier & Scholz, 2012, a parasite of Enhydris enhydris (Ophidia: Homalopsidae) in Cambodia. Molecular data confirm a wide distribution of A. shipleyi (isolates from Malaysia and Vietnam were almost identical) and indicate a strict host specificity (oioxeny) of individual species of the genus. Type specimens of four species made it possible to supplement their morphological descriptions. A survey of all species of Acanthotaenia recognised as valid is presented and the following taxonomic changes are proposed: Acanthotaenia pythonis Wahid, 1968 described from the green python, Morelia viridis, in a zoo, is transferred to Kapsulotaenia as Kapsulotaenia pythonis (Wahid, 1968) n. comb., because it possesses intrauterine eggs grouped in capsules. Acanthotaenia gracilis (Beddard, 1913) from Varanus varius in Australia is considered to be species inquirenda because its original descriptions did not contain sufficient data for adequate circumscription and differentiation from congeners and type material was not available. Generic diagnosis of Acanthotaenia is amended and a key to its seven species is provided.
Eukaryotes of the genus Plasmodium cause malaria, a parasitic disease responsible for substantial morbidity and mortality in humans. Yet, the nature and abundance of any viruses carried by these divergent eukaryotic parasites is unknown. We investigated the Plasmodium virome by performing a meta-transcriptomic analysis of blood samples taken from patients suffering from malaria and infected with P. vivax, P. falciparum or P. knowlesi. This resulted in the identification of a narnavirus-like sequence, encoding an RNA polymerase and restricted to P. vivax samples, as well as an associated viral segment of unknown function. These data, confirmed by PCR, are indicative of a novel RNA virus that we term Matryoshka RNA virus 1 (MaRNAV-1) to reflect its analogy to a "Russian doll": a virus, infecting a parasite, infecting an animal. Additional screening revealed that MaRNAV-1 was abundant in geographically diverse P. vivax derived from humans and mosquitoes, strongly supporting its association with this parasite, and not in any of the other Plasmodium samples analyzed here nor Anopheles mosquitoes in the absence of Plasmodium. Notably, related bi-segmented narnavirus-like sequences (MaRNAV-2) were retrieved from Australian birds infected with a Leucocytozoon-a genus of eukaryotic parasites that group with Plasmodium in the Apicomplexa subclass hematozoa. Together, these data support the establishment of two new phylogenetically divergent and genomically distinct viral species associated with protists, including the first virus likely infecting Plasmodium parasites. As well as broadening our understanding of the diversity and evolutionary history of the eukaryotic virosphere, the restriction to P. vivax may be of importance in understanding P. vivax-specific biology in humans and mosquitoes, and how viral co-infection might alter host responses at each stage of the P. vivax life-cycle.