The mitochondrion of Plasmodium falciparum is required for cellular acetyl-CoA metabolism and protein acetylation.

Coenzyme A (CoA) biosynthesis is an excellent target for antimalarial intervention. While most studies have focused on the use of CoA to produce acetyl-CoA in the apicoplast and the cytosol of malaria parasites, mitochondrial acetyl-CoA production is less well understood. In the current study, we performed metabolite-labeling experiments to measure endogenous metabolites in Plasmodium falciparum lines with genetic deletions affecting mitochondrial dehydrogenase activity. Our results show that the mitochondrion is required for cellular acetyl-CoA biosynthesis and identify a synthetic lethal relationship between the two main ketoacid dehydrogenase enzymes. The activity of these enzymes is dependent on the lipoate attachment enzyme LipL2, which is essential for parasite survival solely based on its role in supporting acetyl-CoA metabolism. We also find that acetyl-CoA produced in the mitochondrion is essential for the acetylation of histones and other proteins outside of the mitochondrion. Taken together, our results demonstrate that the mitochondrion is required for cellular acetyl-CoA metabolism and protein acetylation essential for parasite survival.

Knowledge, compliance, and challenges in anti-malarial products usage: a systematic review of at-risk communities for zoonotic malaria.

BACKGROUND: Zoonotic malaria is a growing public health threat in the WHO Southeast Asia (SEA) and Western Pacific (WP) regions. Despite vector-control measures, the distribution of Macaque fascicularis and M. nemestrina, and Anopheles mosquitoes carrying non-human simian malaria parasites poses challenges to malaria elimination. The systematic review assesses the literature on knowledge and malaria-preventive practices in zoonotic malaria-affected areas across the WHO SEA and WP, aiming to identify challenges for malaria control. METHODS: Peer-reviewed articles published in English, Malay and Indonesian between January 2010 and December 2022 were searched in OVID Medline, Scopus, Web of Science, and Google Scholar. Studies of any design-excluding reviews, conference proceedings, and reports from all WHO SEA and WP countries vulnerable to zoonotic malaria-were included. Backwards-reference screening and thematic analysis were conducted. RESULTS: Among 4,174 initially searched articles, 22 peer-reviewed articles met the inclusion criteria. An additional seven articles were identified through backwards-reference screening, resulting in a total of 29 articles for this review. Half of these studies were conducted in Cambodia, Myanmar, Malaysia, and Thailand, mainly in forests and remote communities. The review highlighted inconsistencies in the operationalization of knowledge, and five major themes were identified related to knowledge: causation and transmission, symptoms, treatment, severity and complications, and malaria prevention. While participants generally had some understanding of malaria causation/transmission, minority and indigenous ethnic groups demonstrated limited knowledge and held misconceptions, such as attributing malaria to drinking dirty water. Preventive practices included traditional and non-traditional or modern methods-with a preference for traditional approaches to avoid mosquito bites. Challenges to malaria control included feasibility, cost, and access to healthcare services. CONCLUSION: This review provides insights into knowledge, local understandings, and preventive practices related to malaria in the WHO SEA and WP regions. The findings highlight the need for future research to explore the knowledge of at-risk communities regarding zoonotic malaria, their perceive threat of the disease and factors exposing them to zoonotic malaria. New strategies must be developed for zoonotic malaria programs tailored to local contexts, emphasizing the significance of community participation, health education, and socio-behavioural change initiatives. It is important to consider the interconnectedness of human health, environmental and non-human primates conservation. Socio-cultural nuances should also be carefully considered in the design and implementation of these programs to ensure their effect tailored to local contexts.

Blood meal profile and positivity rate with malaria parasites among different malaria vectors in Sudan.

BACKGROUND: Malaria is a life-threatening public health problem globally with particularly heavy burden in the sub-Saharan Africa including Sudan. The understanding of feeding preference of malaria vectors on different hosts is a major challenge for hindering the transmission cycle of malaria. In this study, blood meals taken by blood-fed Anopheles mosquitoes collected from the field in malaria endemic areas of Sudan were analysed for source of blood meal and malaria parasite presence. METHODS: Anopheles mosquitoes were collected from different regions in Sudan: Khartoum state, Sennar state, Northern state, and El Gedarif state between September 2020 and February 2021. Anopheles mosquitoes were collected using the standard pyrethrum spray catch and back-pack aspirator. Mosquito samples were sorted and morphologically identified to species level using international identification keys. Morphologically identified mosquito species were also confirmed using PCR. Genomic DNA was extracted from mosquitoes for molecular identification of blood meal source and parasite detection. The presence of Plasmodium species DNA in each mosquito sample was investigated using semi-nested PCR. Frequency of each blood meal source, Anopheles mosquito vector, and malaria parasite detected was calculated. Positivity rate of each fed female Anopheles mosquito was calculated for each species. RESULTS: A total of 2132 Anopheles mosquitoes were collected. 571 (26.8%) were males and 1561 (73.2%) were females classified based on their abdominal status into 1048 (67.1%) gravid, 274 (17.6%) fed, and 239 (15.3%) unfed females. Among the blood fed Anopheles mosquitoes, 263 (96.0%) were morphologically identified and confirmed using PCR to Anopheles arabiensis, 9 (3.3%) to Anopheles stephensi, and 2 (0.7%) to Anopheles rufipes. Of 274 blood-fed An. arabiensis, 68 (25.9%) fed on mixed blood meals from human and cattle, 8 (3.0%) fed on cattle and goat, and 13 (4.8%) fed on human, cattle and goat. For single blood meal sources, 70 (26.6%) fed on human, 95 (36.1%) fed on cattle, 8 (3.0%) fed on goat, and 1 (0.4%) fed on dog. While An. rufipes and An. stephensi fed on dog (2; 0.75%) and cattle (9; 3.3%), respectively. Plasmodium parasite detection in the blood meals showed that 25/274 (9.1%) An. arabiensis meals were positive for Plasmodium vivax and 19/274 (6.9%) An. arabiensis meals were positive for Plasmodium falciparum. The rate of positivity of An. arabiensis with any Plasmodium species was 16.7%. However, the positivity rate with P. falciparum only was 7.2%, while P. vivax was 9.5%. Both An. rufipes and An. stephensi were having positivity rates of 0.0% each. CONCLUSIONS: This study which was mainly on blood-fed Anopheles mosquitoes showed a diversity in the type of diet from human, cattle, and goat. Anopheles mosquitoes especially An. arabiensis in Sudan, are opportunistic blood feeders and can feed broadly on both human and cattle. The application of blood meal identification is not only important in malaria vector epidemiological surveillance but also is very useful in areas where arthropods exhibit zoophilic feeding behaviour for mammals.

Plasmodium vinckei genomes provide insights into the pan-genome and evolution of rodent malaria parasites.

BACKGROUND: Rodent malaria parasites (RMPs) serve as tractable tools to study malaria parasite biology and host-parasite-vector interactions. Among the four RMPs originally collected from wild thicket rats in sub-Saharan Central Africa and adapted to laboratory mice, Plasmodium vinckei is the most geographically widespread with isolates collected from five separate locations. However, there is a lack of extensive phenotype and genotype data associated with this species, thus hindering its use in experimental studies. RESULTS: We have generated a comprehensive genetic resource for P. vinckei comprising of five reference-quality genomes, one for each of its subspecies, blood-stage RNA sequencing data for five P. vinckei isolates, and genotypes and growth phenotypes for ten isolates. Additionally, we sequenced seven isolates of the RMP species Plasmodium chabaudi and Plasmodium yoelii, thus extending genotypic information for four additional subspecies enabling a re-evaluation of the genotypic diversity and evolutionary history of RMPs. The five subspecies of P. vinckei have diverged widely from their common ancestor and have undergone large-scale genome rearrangements. Comparing P. vinckei genotypes reveals region-specific selection pressures particularly on genes involved in mosquito transmission. Using phylogenetic analyses, we show that RMP multigene families have evolved differently across the vinckei and berghei groups of RMPs and that family-specific expansions in P. chabaudi and P. vinckei occurred in the common vinckei group ancestor prior to speciation. The erythrocyte membrane antigen 1 and fam-c families in particular show considerable expansions among the lowland forest-dwelling P. vinckei parasites. The subspecies from the highland forests of Katanga, P. v. vinckei, has a uniquely smaller genome, a reduced multigene family repertoire and is also amenable to transfection making it an ideal parasite for reverse genetics. We also show that P. vinckei parasites are amenable to genetic crosses. CONCLUSIONS: Plasmodium vinckei isolates display a large degree of phenotypic and genotypic diversity and could serve as a resource to study parasite virulence and immunogenicity. Inclusion of P. vinckei genomes provide new insights into the evolution of RMPs and their multigene families. Amenability to genetic crossing and transfection make them also suitable for classical and functional genetics to study Plasmodium biology.

Mathematical assessment of the impact of human-antibodies on sporogony during the within-mosquito dynamics of Plasmodium falciparum parasites.

We develop and analyze a deterministic ordinary differential equation mathematical model for the within-mosquito dynamics of the Plasmodium falciparum malaria parasite. Our model takes into account the action and effect of blood resident human-antibodies, ingested by the mosquito during a blood meal from humans, in inhibiting gamete fertilization. The model also captures subsequent developmental processes that lead to the different forms of the parasite within the mosquito. Continuous functions are used to model the switching transition from oocyst to sporozoites as well as human antibody density variations within the mosquito gut are proposed and used. In sum, our model integrates the developmental stages of the parasite within the mosquito such as gametogenesis, fertilization and sporogenesis culminating in the formation of sporozoites. Quantitative and qualitative analyses including a sensitivity analysis for influential parameters are performed. We quantify the average sporozoite load produced at the end of the within-mosquito malaria parasite's developmental stages. Our analysis shows that an increase in the efficiency of the ingested human antibodies in inhibiting fertilization within the mosquito's gut results in lowering the density of oocysts and hence sporozoites that are eventually produced by each mosquito vector. So, it is possible to control and limit oocysts development and hence sporozoites development within a mosquito by boosting the efficiency of antibodies as a pathway to the development of transmission-blocking vaccines which could potentially reduce oocysts prevalence among mosquitoes and hence reduce the transmission potential from mosquitoes to human.

Plasmodium transmission differs between mosquito species and parasite lineages.

Factors such as the particular combination of parasite-mosquito species, their co-evolutionary history and the host's parasite load greatly affect parasite transmission. However, the importance of these factors in the epidemiology of mosquito-borne parasites, such as avian malaria parasites, is largely unknown. Here, we assessed the competence of two mosquito species [Culex pipiens and Aedes (Ochlerotatus) caspius], for the transmission of four avian Plasmodium lineages (Plasmodium relictum SGS1 and GRW11 and Plasmodium cathemerium-related lineages COLL1 and PADOM01) naturally infecting wild house sparrows. We assessed the effects of parasite identity and parasite load on Plasmodium transmission risk through its effects on the transmission rate and mosquito survival. We found that Cx. pipiens was able to transmit the four Plasmodium lineages, while Ae. caspius was unable to transmit any of them. However, Cx. pipiens mosquitoes fed on birds infected by P. relictum showed a lower survival and transmission rate than those fed on birds infected by parasites related to P. cathemerium. Non-significant associations were found with the host-parasite load. Our results confirm the existence of inter- and intra-specific differences in the ability of Plasmodium lineages to develop in mosquito species and their effects on the survival of mosquitoes that result in important differences in the transmission risk of the different avian malaria parasite lineages studied.

Relationship between blood Lead status and anemia in Ugandan children with malaria infection.

BACKGROUND: In Uganda, childhood anemia remains a health challenge and is associated with malaria infection as well as iron deficiency. Iron deficiency is intertwined with nutritional status, age and other comorbidities including helminths and Lead toxicity. Environmental Lead levels accounts for one's blood Lead (BL) levels. Blood Lead competitively blocks iron absorption, inhibits hemoglobin (Hb) biosynthesis and elevates free erythrocyte protoporphyrin (FEP) levels. Lead toxicity's contribution towards anemia pathogenesis, especially during malaria infection has not been studied. Concomitant exposure to both malaria infection and Lead pollution, exacerbates the anemia status. This study therefore aimed at expounding the anemia status of these Ugandan children aged under 5years who are exposed to both malaria infection and environmental Lead pollution. METHODS: Briefly, venous blood samples from 198 children were microscopically assayed for malaria parasite density (PD), and hemoglobin (Hb) concentrations using the cyanmethemoglobin method, while BL and FEP levels were determined by the standard atomic absorption spectrophotometric and fluorometric methods respectively. RESULTS: One hundred and fifty-one (76.3%) of the children analyzed had moderate anemia (Hb <10>5 g/dL) with Means of BLL=8.6 µg/dL, Hb =7.5 g/dL, FEP/Hb =8.3 µg/g and PD =3.21×103 parasites / µL, while eight (4%) were severely anemic (<5 g/dL). Regression analysis and statistical correlation between PD and Hb (r = -0.231, R2= 0.15 P-value < 0.001) was negative and weak as compared to that between FEP/Hb and Hb (r = -0.6, R2=0.572 P-value=0.001). CONCLUSION: Based on the study's findings, we conclude that BL significantly contributes to the pathogenesis of anemia and therefore its co-existence with malaria infection in the host exacerbates the anemia status.

Real-time Malaria Parasite Screening in Thick Blood Smears for Low-Resource Setting.

Malaria is a serious public health problem in many parts of the world. Early diagnosis and prompt effective treatment are required to avoid anemia, organ failure, and malaria-associated deaths. Microscopic analysis of blood samples is the preferred method for diagnosis. However, manual microscopic examination is very laborious and requires skilled health personnel of which there is a critical shortage in the developing world such as in sub-Saharan Africa. Critical shortages of trained health personnel and the inability to cope with the workload to examine malaria slides are among the main limitations of malaria microscopy especially in low-resource and high disease burden areas. We present a low-cost alternative and complementary solution for rapid malaria screening for low resource settings to potentially reduce the dependence on manual microscopic examination. We develop an image processing pipeline using a modified YOLOv3 detection algorithm to run in real time on low-cost devices. We test the performance of our solution on two datasets. In the dataset collected using a microscope camera, our model achieved 99.07% accuracy and 97.46% accuracy on the dataset collected using a mobile phone camera. While the mean average precision of our model is on par with human experts at an object level, we are several orders of magnitude faster than human experts as we can detect parasites in images as well as videos in real time.

Solution Structures and Dynamic Assembly of the 24-Meric Plasmodial Pdx1-Pdx2 Complex.

Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host's metabolic processes or even absent in the host. In this context, the essential presence of vitamin B6 biosynthesis enzymes in Plasmodium, the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium vivax PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1-Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.

A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites.

BACKGROUND: The transcriptional regulation that occurs in malaria parasites during the erythrocytic stages of infection can be studied in vivo with rodent malaria parasites propagated in mice. Time-series transcriptome profiling commonly involves the euthanasia of groups of mice at specific time points followed by the extraction of parasite RNA from whole blood samples. Current methodologies for parasite RNA extraction involve several steps and when multiple time points are profiled, these protocols are laborious, time-consuming, and require the euthanization of large cohorts of mice. RESULTS: A simplified protocol has been designed for parasite RNA extraction from blood volumes as low as 20 µL (microsamples), serially bled from mice via tail snips and directly lysed with TRIzol reagent. Gene expression data derived from microsampling using RNA-seq were closely matched to those derived from larger volumes of leucocyte-depleted and saponin-treated blood obtained from euthanized mice with high reproducibility between biological replicates. Transcriptome profiling of microsamples taken at different time points during the intra-erythrocytic developmental cycle of the rodent malaria parasite Plasmodium vinckei revealed the transcriptional cascade commonly observed in malaria parasites. CONCLUSIONS: Microsampling is a quick, robust and cost-efficient approach to sample collection for in vivo time-series transcriptomic studies in rodent malaria parasites.

Evolutionary lability of host associations promotes phylogenetic overdispersion of co-infecting blood parasites.

Co-infections with multiple parasite taxa are ubiquitous in nature and have the potential to impact the co-evolutionary dynamics between host and parasite, though patterns of phylogenetic community structure of co-infecting parasites and the processes that generate these patterns have rarely been studied across diverse host-parasite communities. Here, we tested for the roles of host and parasite evolutionary history as well as environmental variables as drivers of phylogenetic community structure among co-infecting haemosporidian (malaria) parasites and their avian hosts in the North American boreal forest, a region characterized by an extraordinarily high blood parasite co-infection rate. We used multiple methods to identify non-random patterns of co-infection among parasite species and determined whether these patterns were influenced more by co-evolutionary host associations or environmental variables. We used model-based approaches to test whether parasites that occurred together in a single host individual exhibited phylogenetic clustering or overdispersion. Lastly, we tested whether the observed phylogenetic community structure could be explained by parasites having convergently evolved similar patterns of host associations. We found that haemosporidian parasite co-infections occurred at a high frequency in the boreal forest system and that parasite taxa co-occurred in significantly non-random patterns within host individuals and among host species. Parasite taxa that occurred in co-infections tended to be phylogenetically overdispersed. We show that this pattern of phylogenetic overdispersion can be attributed largely to the effect of evolutionarily labile, convergent host associations that have resulted in the pool of parasites that have the potential to infect a given host consisting nearly exclusively of distantly related lineages. Our findings illustrate that environmental filtering of convergent traits can produce phylogenetically overdispersed communities, even at the level of co-infecting parasites within an individual host organism. Broadly, this analysis illustrates how co-evolutionary history can have a strong influence on the modern phylogenetic community assembly of diverse host-symbiont communities.

Melatonin activates FIS1, DYN1, and DYN2 Plasmodium falciparum related-genes for mitochondria fission: Mitoemerald-GFP as a tool to visualize mitochondria structure.

Malaria causes millions of deaths worldwide and is considered a huge burden to underdeveloped countries. The number of cases with resistance to all antimalarials is continuously increasing, making the identification of novel drugs a very urgent necessity. A potentially very interesting target for novel therapeutic intervention is the parasite mitochondrion. In this work, we studied in Plasmodium falciparum 3 genes coding for proteins homologues of the mammalian FIS1 (Mitochondrial Fission Protein 1) and DRP1 (Dynamin Related Protein 1) involved in mitochondrial fission. We studied the expression of P. falciparum genes that show ample sequence and structural homologies with the mammalian counterparts, namely FIS1, DYN1, and DYN2. The encoded proteins are characterized by a distinct pattern of expression throughout the erythrocytic cycle of P. falciparum, and their mRNAs are modulated by treating the parasite with the host hormone melatonin. We have previously reported that the knockout of the Plasmodium gene that codes for protein kinase 7 is essential for melatonin sensing. We here show that PfPk7 knockout results in major alterations of mitochondrial fission genes expression when compared to wild-type parasites, and no change in fission proteins expression upon treatment with the host hormone. Finally, we have compared the morphological characteristics (using MitoTracker Red CMX Ros) and oxygen consumption properties of P. falciparum mitochondria in wild-type parasites and PfPk7 Knockout strains. A novel GFP construct targeted to the mitochondrial matrix to wild-type parasites was also developed to visualize P. falciparum mitochondria. We here show that, the functional characteristics of P. falciparum are profoundly altered in cells lacking protein kinase 7, suggesting that this enzyme plays a major role in the control of mitochondrial morphogenesis and maturation during the intra-erythrocyte cell cycle progression.

Vaccination with chemically attenuated Plasmodium falciparum asexual blood-stage parasites induces parasite-specific cellular immune responses in malaria-naïve volunteers: a pilot study.

BACKGROUND: The continuing morbidity and mortality associated with infection with malaria parasites highlights the urgent need for a vaccine. The efficacy of sub-unit vaccines tested in clinical trials in malaria-endemic areas has thus far been disappointing, sparking renewed interest in the whole parasite vaccine approach. We previously showed that a chemically attenuated whole parasite asexual blood-stage vaccine induced CD4+ T cell-dependent protection against challenge with homologous and heterologous parasites in rodent models of malaria. METHODS: In this current study, we evaluated the immunogenicity and safety of chemically attenuated asexual blood-stage Plasmodium falciparum (Pf) parasites in eight malaria-naïve human volunteers. Study participants received a single dose of 3 × 107 Pf pRBC that had been treated in vitro with the cyclopropylpyrolloindole analogue, tafuramycin-A. RESULTS: We demonstrate that Pf asexual blood-stage parasites that are completely attenuated are immunogenic, safe and well tolerated in malaria-naïve volunteers. Following vaccination with a single dose, species and strain transcending Plasmodium-specific T cell responses were induced in recipients. This included induction of Plasmodium-specific lymphoproliferative responses, T cells secreting the parasiticidal cytokines, IFN-γ and TNF, and CD3+CD45RO+ memory T cells. Pf-specific IgG was not detected. CONCLUSIONS: This is the first clinical study evaluating a whole parasite blood-stage malaria vaccine. Following administration of a single dose of completely attenuated Pf asexual blood-stage parasites, Plasmodium-specific T cell responses were induced while Pf-specific antibodies were not detected. These results support further evaluation of this chemically attenuated vaccine in humans. TRIAL REGISTRATION: Trial registration: ACTRN12614000228684 . Registered 4 March 2014.

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine.

Hematin crystallization is the primary mechanism of heme detoxification in malaria parasites and the target of the quinoline class of antimalarials. Despite numerous studies of malaria pathophysiology, fundamental questions regarding hematin growth and inhibition remain. Among them are the identity of the crystallization medium in vivo, aqueous or organic; the mechanism of crystallization, classical or nonclassical; and whether quinoline antimalarials inhibit crystallization by sequestering hematin in the solution, or by blocking surface sites crucial for growth. Here we use time-resolved in situ atomic force microscopy (AFM) and show that the lipid subphase in the parasite may be a preferred growth medium. We provide, to our knowledge, the first evidence of the molecular mechanisms of hematin crystallization and inhibition by chloroquine, a common quinoline antimalarial drug. AFM observations demonstrate that crystallization strictly follows a classical mechanism wherein new crystal layers are generated by 2D nucleation and grow by the attachment of solute molecules. We identify four classes of surface sites available for binding of potential drugs and propose respective mechanisms of drug action. Further studies reveal that chloroquine inhibits hematin crystallization by binding to molecularly flat {100} surfaces. A 2-µM concentration of chloroquine fully arrests layer generation and step advancement, which is ∼10(4)× less than hematin's physiological concentration. Our results suggest that adsorption at specific growth sites may be a general mode of hemozoin growth inhibition for the quinoline antimalarials. Because the atomic structures of the identified sites are known, this insight could advance the future design and/or optimization of new antimalarials.

Absence of haemosporidian parasite infections in the long-lived Cory's shearwater: evidence from molecular analyses and review of the literature.

The apparent scarcity or absence of blood parasites in some avian groups, such as seabirds, has been related to intrinsic and extrinsic factors including host immunological capacity, host-parasite assemblage, and ecological parameters, but also to reduced sensitivity of some methods to detect low parasite prevalence/intensities of infection. Here, we examined the haemosporidian parasite prevalence in a breeding population of Cory's shearwater Calonectris diomedea borealis, a long-distance migrant seabird, nesting in the Macaronesian region, in the Eastern Atlantic. Previous studies on Calonectris diomedea complex were based on small sample sizes providing weak evidence for a lack of infections by haemoparasites. Here, we investigated the presence of both parasite infections in C. d. borealis and larvae of potential mosquito vectors on the area. By employing a PCR-based assay, we extensively examined the prevalence of blood parasites belonging to the genera Plasmodium, Haemoproteus, and Leucocytozoon in 286 individuals from different life stages (i.e., chicks, immatures, sabbatical, and breeding adults), facing their specific energetic trade-offs (immunological functions vs. life history activities). We sampled immatures and adult shearwaters, of different sexes, ages, and migratory origins, from two sub-colonies. None of the sampled individuals were infected by these parasites, supporting the hypothesis that there was no in situ or ex situ transmission of vector-borne parasites in marine habitats irrespective of host's life stage and in spite of the presence of the potential Plasmodium vector Culiseta longiareolata breeding in the area. These results suggest that the lack of transmission of haemosporidian parasites on Selvagem Grande may be related to the lack of suitable dipteran vectors at the study sites, which may result from the geographic isolation of this area.

Rarity of Mixed Species Malaria with Plasmodium falciparum and Plasmodium malariae in Travelers to Saarland in Germany.

Malaria is an acute, life-threatening infectious disease that spreads in tropical and subtropical regions. Malaria is mainly brought over to Germany by travelers, so the disease can be overlooked due to its nonspecific symptoms and a lack of experience of attending physicians. The aim of this study was to analyze, retrospectively, epidemiological and clinical data from patients examined for malaria. Patient data were collected from hospital charts at the Department of Internal Medicine, Saarland University Medical Center, Germany, for the period of 2004-2012. The data of patients with and without malaria were compared in terms of their epidemiological, demographic, clinical, and medical treatment aspects. We identified found 15 patients with malaria (28.3 %, mean age 42.3 ± 16.5 years, three females [20 %]; 95 % confidence interval of 0.2-0.4) out of the 53 patients examined. Mainly locals brought malaria over to Homburg, Germany (p = 0.009). Malaria tropica was the most common species (p < 0.0001). One patient (6.7 %) with malaria, who had recently traveled, had a mixed infection of Plasmodium falciparum and Plasmodium malariae (p = 0.670). Malaria is characterized by thrombocytopenia (p = 0.047) and elevated C-reactive protein (p = 0.019) in serum, and fever is the leading symptom (p = 0.031). In most cases, malaria was brought from Ghana (33.3 %). Further, patients had contracted malaria despite malaria prophylaxis (33.3 %, p = 0.670). In conclusion, malaria test should be used in patients with fever after a journey from Africa. Malaria caused by Plasmodium falciparum is the most common species of brought over malaria. Mixed-species Plasmodium falciparum and Plasmodium malariae are uncommon in travelers with malaria.

The rediscovery of malaria parasites of ungulates.

Over a hundred years since their first description in 1913, the sparsely described malaria parasites (genus Plasmodium) of ungulates have been rediscovered using molecular typing techniques. In the span of weeks, three studies have appeared describing the genetic characterization and phylogenetic analyses of malaria parasites from African antelope (Cephalophus spp.) and goat (Capra aegagrus hircus), Asian water buffalo (Bubalus bubalis), and North American white-tailed deer (Odocoileus virginianus). Here we unify the contributions from those studies with the literature on pre-molecular characterizations of ungulate malaria parasites, which are largely based on surveys of Giemsa-reagent stained blood smears. We present a phylogenetic tree generated from all available ungulate malaria parasite sequence data, and show that parasites from African duiker antelope and goat, Asian water buffalo and New World white-tailed deer group together in a clade, which branches early in Plasmodium evolution. Anopheline mosquitoes appear to be the dominant, if not sole vectors for parasite transmission. We pose questions for future phylogenetic studies, and discuss topics that we hope will spur further molecular and cellular studies of ungulate malaria parasites.

The conserved clag multigene family of malaria parasites: essential roles in host-pathogen interaction.

The clag multigene family is strictly conserved in malaria parasites, but absent from neighboring genera of protozoan parasites. Early research pointed to roles in merozoite invasion and infected cell cytoadherence, but more recent studies have implicated channel-mediated uptake of ions and nutrients from host plasma. Here, we review the current understanding of this gene family, which appears to be central to host-parasite interactions and an important therapeutic target.

Can hemozoin alone cause host anaemia?

Both schistosomes and malaria parasites produce hemozoin and cause host anaemia. However, the relationship between anaemia and hemozoin is unclear. Although some studies have proposed that hemozoin is related to anaemia in malaria patients, whether hemozoin alone can cause anaemia in patients infected by malaria parasites or schistosomes is uncertain. To investigate the effect of hemozoin on hosts, ß-haematin was injected intravenously to normal mice. Then, liver and spleen tissues were observed. Mouse blood was examined. Red blood cells (RBCs), white blood cells (WBCs) and haemoglobin were analysed. Macrophage changes in the spleens and marrow cells were compared using immunofluorescence and H&E or Giemsa stain, respectively. We found that after 15 injections of ß-haematin, a large amount of ß-haematin was observed to deposit in the livers and spleens. Splenomegaly and bone marrow mild hyperplasia were detected. The average number of RBCs, average number of WBCs and average concentration of haemoglobin decreased significantly from 9.36 × 1012 cells/L to 8.7 × 1012 cells/L, 3.8 × 109 cells/L to 1.7 × 109 cells/L and 142.8 g/L to 131.8 g/L, respectively. In specific, the number of macrophages in the spleens greatly increased after ß-haematin infection. The results showed that injections of ß-haematin alone can cause anaemia possibly through hypersplenism.

Computational microscopic imaging for malaria parasite detection: a systematic review.

Malaria, being an epidemic disease, demands its rapid and accurate diagnosis for proper intervention. Microscopic image-based characterization of erythrocytes plays an integral role in screening of malaria parasites. In practice, microscopic evaluation of blood smear image is the gold standard for malaria diagnosis; where the pathologist visually examines the stained slide under the light microscope. This visual inspection is subjective, error-prone and time consuming. In order to address such issues, computational microscopic imaging methods have been given importance in recent times in the field of digital pathology. Recently, such quantitative microscopic techniques have rapidly evolved for abnormal erythrocyte detection, segmentation and semi/fully automated classification by minimizing such diagnostic errors for computerized malaria detection. The aim of this paper is to present a review on enhancement, segmentation, microscopic feature extraction and computer-aided classification for malaria parasite detection.