Transcriptional analysis of an immune-responsive serine protease from Indian malarial vector, <Emphasis Type="Italic">Anopheles culicifacies</Emphasis>

Background  

The main vector for transmission of malaria in India is the Anopheles culicifacies mosquito species, a naturally selected subgroup of which is completely refractory (R) to transmission of the malaria parasite, Plasmodium vivax;     

The molecular evolution of four anti-malarial immune genes in the <Emphasis Type="Italic">Anopheles gambiae</Emphasis> species complex

Background  

If the insect innate immune system is to be used as a potential blocking step in transmission of malaria, then it will require targeting one or a few genes with highest relevance and ease of manipulation. The problem is to identify and manipulate those of most importance to malaria infection without the risk of decreasing the mosquito's ability to stave off infections by microbes in general. Molecular evolution methodologies and concepts can help identify such genes. Within the setting of a comparative molecular population genetic and phylogenetic framework, involving six species of the Anopheles gambiae complex, we investigated whether a set of four pre-selected immunity genes (gambicin, NOS, Rel2 and FBN9) might have evolved under selection pressure imposed by the malaria parasite.     

Preparation of pure and intact <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> plasma membrane vesicles and partial characterisation of the plasma membrane ATPase

Background  

In host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact Plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport.     

Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

Background

The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites.

Methods

Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively.

Results

Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite.

Conclusions

Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host.     

A theoretical approach to predicting the success of genetic manipulation of malaria mosquitoes in malaria control

Background  

Mosquitoes that have been genetically modified to better encapsulate the malaria parasite Plasmodium falciparum are being considered as a possible tool in the control of malaria. Hopes for this have been raised with the identification of genes involved in the encapsulation response and with advances in the tools required to transform mosquitoes. However, we have only very little understanding of the conditions that would allow such genes to spread in natural populations.     

Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases

Background  

Purine nucleoside phosphorylase (PNP) is central to purine salvage mechanisms in Plasmodium parasites, the causative agents of malaria. Most human malaria results from infection either by Plasmodium falciparum (Pf), the deadliest form of the parasite, or by the widespread Plasmodium vivax (Pv). Whereas the PNP enzyme from Pf has previously been studied in detail, despite the prevalence of Pv little is known about many of the key metabolic enzymes from this parasite, including PvPNP.     

A deep sequencing approach to estimate <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> complexity of infection (COI) and explore apical membrane antigen 1 diversity

Background

Humans living in regions with high falciparum malaria transmission intensity harbour multi-strain infections comprised of several genetically distinct malaria haplotypes. The number of distinct malaria parasite haplotypes identified from an infected human host at a given time is referred to as the complexity of infection (COI). In this study, an amplicon-based deep sequencing method targeting the Plasmodium falciparum apical membrane antigen 1 (pfama1) was utilized to (1) investigate the relationship between P. falciparum prevalence and COI, (2) to explore the population genetic structure of P. falciparum parasites from malaria asymptomatic individuals participating in the 2007 Demographic and Health Survey (DHS) in the Democratic Republic of Congo (DRC), and (3) to explore selection pressures on geospatially divergent parasite populations by comparing AMA1 amino acid frequencies in the DRC and Mali.

Results

A total of 900 P. falciparum infections across 11 DRC provinces were examined. Deep sequencing of both individuals, for COI analysis, and pools of individuals, to examine population structure, identified 77 unique pfama1 haplotypes. The majority of individual infections (64.5%) contained polyclonal (COI > 1) malaria infections based on the presence of genetically distinct pfama1 haplotypes. A minimal correlation between COI and malaria prevalence as determined by sensitive real-time PCR was identified. Population genetic analyses revealed extensive haplotype diversity, the vast majority of which was shared across the sites. AMA1 amino acid frequencies were similar between parasite populations in the DRC and Mali.

Conclusions

Amplicon-based deep sequencing is a useful tool for the detection of multi-strain infections that can aid in the understanding of antigen heterogeneity of potential malaria vaccine candidates, population genetics of malaria parasites, and factors that influence complex, polyclonal malaria infections. While AMA1 and other diverse markers under balancing selection may perform well for understanding COI, they may offer little geographic or temporal discrimination between parasite populations.

     

Roll Back Malaria? The scarcity of international aid for malaria control

Background

Intraerythrocytic malaria parasites actively import obligate nutrients from serum and export proteins and lipids to erythrocyte cytoplasm and membrane. The import of macromolecules in the malaria parasite has been the subject of many debates. To understand the import of macromolecules by the parasite, we studied the uptake of proteins by Plasmodium falciparum infected human erythrocyte.

Methods

Proteins were biotin labelled individually, purified on a gel filtration column and added to uninfected and infected asynchronized culture. The uptake of these proteins by malaria parasites was determined by western blot analysis of parasite pellet and their different fractions using streptavidin-horseradish conjugate. To further confirm this import, we studied the uptake of¹²⁵I-labelled proteins by western blot analysis as well as used direct immunofluorescence method.

Results

Here we show that biotin labelled and radio-iodinated polypeptides of molecular sizes in the range of 45 to 206 kDa, when added in the culture medium, get direct access to the parasite membrane through a membrane network by by-passing the erythrocyte cytosol. The import of these polypeptides is ATP-dependent as sodium azide treatment blocks this uptake. We also show that malaria parasites have the ability to take up and degrade biotin labelled human serum albumin, which has been shown to be essential for the parasite growth.

Conclusions

These results can be used, as a basis to explore the role of human serum albumin in the intraerythrocytic development of parasites, and this in turn can be an important adjunct to the development of novel antimalarial drugs.     

Malaria prophylaxis policy for travellers from Europe to the Indian Sub Continent

Background

Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge.

Methods

The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia.

Results

Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities.

Conclusion

It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density.     

Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites

Background  

The apicomplexan parasite Plasmodium falciparum causes the most severe form of malaria in humans. After invasion into erythrocytes, asexual parasite stages drastically alter their host cell and export remodeling and virulence proteins. Previously, we have reported identification and functional analysis of a short motif necessary for export of proteins out of the parasite and into the red blood cell.     

Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax

Background  

Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites.     

Antipyretic effect of ibuprofen in Gabonese children with uncomplicated falciparum malaria: a randomized, double-blind, placebo-controlled trial

Background

The protection afforded by human erythrocyte polymorphisms against the malaria parasite, Plasmodium falciparum, has been proposed to be due to reduced ability of the parasite to invade or develop in erythrocytes. If this were the case, variable levels of parasitaemia and rates of seroconversion to infected-erythrocyte variant surface antigens (VSA) should be seen in different host genotypes.

Methods

To test this hypothesis, P. falciparum parasitaemia and anti-VSA antibody levels were measured in a cohort of 555 asymptomatic children from an area of intense malaria transmission in Papua New Guinea. Linear mixed models were used to investigate the effect of α⁺-thalassaemia, complement receptor-1 and south-east Asian ovalocytosis, as well as glucose-6-phosphate dehydrogenase deficiency and ABO blood group on parasitaemia and age-specific seroconversion to VSA.

Results

No host polymorphism showed a significant association with both parasite prevalence/density and age-specific seroconversion to VSA.

Conclusion

Host erythrocyte polymorphisms commonly found in Papua New Guinea do not effect exposure to blood stage P. falciparum infection. This contrasts with data for sickle cell trait and highlights that the above-mentioned polymorphisms may confer protection against malaria via distinct mechanisms.     

Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites

Background information

The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized.

Results

Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt‐interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab‐interactome using putative parasite Rab‐effectors identified by homology to Ypt‐effectors. We demonstrate its potential by wet‐bench testing three predictions; that casein kinase‐1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP‐dependent protein kinase A (PfPKA‐C) is a PfRab5A and PfRab7 effector.

Conclusions

The establishment of a shared set of physical Ypt/PfRab‐effector proteins sheds light on a core set Plasmodium Rab‐interactants shared with yeast. The PfRab‐interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA‐C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab‐recruited kinases potentially play a role in early and late endosome function in malaria parasites.     

Microsatellite DNA Analysis Revealed a Drastic Genetic Change of Plasmodium vivax Population in the Republic of Korea During 2002 and 2003

Background

Vivax malaria was successfully eliminated in the Republic of Korea (South Korea) in the late 1970s, but it was found to have re-emerged from 1993. In order to control malaria and evaluate the effectiveness of malaria controls, it is important to develop a spatiotemporal understanding of the genetic structure of the parasite population. Here, we estimated the population structure and temporal dynamics of the transmission of Plasmodium vivax in South Korea by analyzing microsatellite DNA markers of the parasite.

Methodology/Principal Findings

We analyzed 14 microsatellite DNA loci of the P. vivax genome from 163 South Korean isolates collected from 1994 to 2008. Allelic data were used to analyze linkage disequilibrium (LD), genetic differentiation and population structure, in order to make a detailed estimate of temporal change in the parasite population. The LD analysis showed a gradual decrease in LD levels, while the levels of genetic differentiation between successive years and analysis of the population structure based on the Bayesian approach suggested that a drastic genetic change occurred in the South Korean population during 2002 and 2003.

Conclusions/Significance

Although relapse and asymptomatic parasite carriage might influence the population structure to some extent, our results suggested the continual introduction of P. vivax into South Korea through other parasite population sources. One possible source, particularly during 2002 and 2003, is North Korea. Molecular epidemiology using microsatellite DNA of the P. vivax population is effective for assessing the population structure and temporal dynamics of parasite transmission; information that can assist in the elimination of vivax malaria in endemic areas.     

Wild Anopheles funestus Mosquito Genotypes Are Permissive for Infection with the Rodent Malaria Parasite,Plasmodium berghei

Background

Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development.

Methods

An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae.

Results

P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18–20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections.

Conclusions

The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus.     

Defining the Geographical Range of the Plasmodium knowlesi Reservoir

Background

The simian malaria parasite, Plasmodium knowlesi, can cause severe and fatal disease in humans yet it is rarely included in routine public health reporting systems for malaria and its geographical range is largely unknown. Because malaria caused by P. knowlesi is a truly neglected tropical disease, there are substantial obstacles to defining the geographical extent and risk of this disease. Information is required on the occurrence of human cases in different locations, on which non-human primates host this parasite and on which vectors are able to transmit it to humans. We undertook a systematic review and ranked the existing evidence, at a subnational spatial scale, to investigate the potential geographical range of the parasite reservoir capable of infecting humans.

Methodology/Principal Findings

After reviewing the published literature we identified potential host and vector species and ranked these based on how informative they are for the presence of an infectious parasite reservoir, based on current evidence. We collated spatial data on parasite occurrence and the ranges of the identified host and vector species. The ranked spatial data allowed us to assign an evidence score to 475 subnational areas in 19 countries and we present the results on a map of the Southeast and South Asia region.

Conclusions/Significance

We have ranked subnational areas within the potential disease range according to evidence for presence of a disease risk to humans, providing geographical evidence to support decisions on prevention, management and prophylaxis. This work also highlights the unknown risk status of large parts of the region. Within this unknown category, our map identifies which areas have most evidence for the potential to support an infectious reservoir and are therefore a priority for further investigation. Furthermore we identify geographical areas where further investigation of putative host and vector species would be highly informative for the region-wide assessment.     

Novel ELISA method as exploratory tool to assess immunity induced by radiated attenuated sporozoites to decipher protective immunity

Background

Whole parasite vaccines provide a unique opportunity for dissecting immune mechanisms and identify antigens that are targeted by immune responses which have the potential to mediate sterile protection against malaria infections. The radiation attenuated sporozoite (PfSPZ) vaccine has been considered the gold standard for malaria vaccines because of its unparalleled efficacy. The immunogenicity of this and other vaccines continues to be evaluated by using recombinant proteins or peptides of known sporozoite antigens. This approach, however, has significant limitations by relying solely on a limited number of known pathogen-associated immune epitopes. Using the full range of antigens expressed by the sporozoite will enable the comprehensive immune-profiling of humoral immune responses induced by whole parasite vaccines. To address this challenge, a novel ELISA based on sporozoites was developed.

Results

The SPZ-ELISA method described in this report can be performed with either freshly dissected sporozoites or with cryopreserved sporozoite lysates. The use of a fixative for reproducible coating is not required. The SPZ-ELISA was first validated using monoclonal antibodies specific for CSP and TRAP and then used for the characterization of immune sera from radiation attenuated sporozoite vaccinees.

Conclusion

Applying this simple and highly reproducible approach to assess immune responses induced by malaria vaccines, both recombinant and whole parasite vaccines, (1) will help in the evaluation of immune responses induced by antigenically complex malaria vaccines such as the irradiated SPZ-vaccine, (2) will facilitate and accelerate the identification of immune correlates of protection, and (3) can also be a valuable assessment tool for antigen discovery as well as down-selection of vaccine formulations and, thereby, guide vaccine design.

     

Molecular monitoring of the Leu-164 mutation of dihydrofolate reductase in a highly sulfadoxine/pyrimethamine-resistant area in Africa

Background

Procalcitonin (PCT) is closely correlated with parasite burden and clinical outcome in falciparum malaria. The role of PCT in tertian malaria has not previously been investigated.

Patients and methods

PCT serum levels in 37 patients with tertian malaria were analysed. Clinical and laboratory parameters were assessed and statistically correlated both to the initial PCT levels and during the course of the disease.

Results

PCT levels rose for one day after commencing treatment and declined thereafter. However, there was no significant correlation with parasite burden, clinical parameters, laboratory values, or the presence of semi-immunity. Before treatment, the majority of patients showed normal or slightly elevated PCT levels (< 2.5 ng/ml), but PCT was markedly elevated (4.8 – 47 ng/ml) in one third of the population. The two groups did not differ by any other of the assessed parameters. Thus, while the post-treatment course of PCT resembles falciparum malaria, the lack of correlation between disease severity and even high PCT levels in a large proportion of patients is intriguing.

Conclusions

There is a fundamental difference in the relationship of PCT with tertian malaria not seen in other infectious diseases in which elevated PCT levels have been observed. This suggests distinct pathophysiological pathways in malaria.     

Co-infections of Malaria and Geohelminthiasis in Two Rural Communities of Nkassomo and Vian in the Mfou Health District,Cameroon

Background

Human co-infection with malaria and helmimths is ubiquitous throughout Africa. Nevertheless, its public health significance on malaria severity remains poorly understood.

Methodology/Principal Findings

To contribute to a better understanding of epidemiology and control of this co-infection in Cameroon, a cross-sectional study was carried out to assess the prevalence of concomitant intestinal geohelminthiasis and malaria, and to evaluate its association with malaria and anaemia in Nkassomo and Vian. Finger prick blood specimens from a total of 263 participants aged 1–95 years were collected for malaria microscopy, assessment of haemoglobin levels, and molecular identification of Plasmodium species by PCR. Fresh stool specimens were also collected for the identification and quantification of geohelminths by the Kato-Katz method. The prevalence of malaria, geohelminths, and co-infections were 77.2%, 28.6%, and 22.1%, respectively. Plasmodium falciparum was the only malaria parasite species identified with mean parasite density of 111 (40; 18,800) parasites/µl of blood. The geohelminths found were Ascaris lumbricoides (21.6%) and Trichuris trichiura (10.8%), with mean parasite densities of 243 (24; 3,552) and 36 (24; 96) eggs/gram of faeces, respectively. Co-infections of A. lumbricoides and P. falciparum were the most frequent and correlated positively. While no significant difference was observed on the prevalences of single and co-infections between the two localities, there was a significant difference in the density of A. lumbricoides infection between the two localities. The overall prevalence of anaemia was 42%, with individuals co-infected with T. trichiura and P. falciparum (60%) being the most at risk. While the prevalence of malaria and anaemia were inversely related to age, children aged 5–14 years were more susceptible to geohelminthiasis and their co-infections with malaria.

Conclusion/Significance

Co-existence of geohelminths and malaria parasites in Nkassomo and Vian enhances the occurrence of co-infections, and consequently, increases the risk for anaemia.     

Environmental,geographical and time-related impacts on avian malaria infections in native and introduced populations of house sparrows (Passer domesticus), a globally invasive species

Aim

The increasing spread of vector-borne diseases has resulted in severe health concerns for humans, domestic animals and wildlife, with changes in land use and the introduction of invasive species being among the main possible causes for this increase. We explored several ecological drivers potentially affecting the local prevalence and richness of avian malaria parasite lineages in native and introduced house sparrows (Passer domesticus) populations.

Location

Global.

Time period

2002–2019.

Major taxa studied

Avian Plasmodium parasites in house sparrows.

Methods

We analysed data from 2,220 samples from 69 localities across all continents, except Antarctica. The influence of environment (urbanization index and human density), geography (altitude, latitude, hemisphere) and time (bird breeding season and years since introduction) were analysed using generalized additive mixed models (GAMMs) and random forests.

Results

Overall, 670 sparrows (30.2%) were infected with 22 Plasmodium lineages. In native populations, parasite prevalence was positively related to urbanization index, with the highest prevalence values in areas with intermediate urbanization levels. Likewise, in introduced populations, prevalence was positively associated with urbanization index; however, higher infection occurred in areas with either extreme high or low levels of urbanization. In introduced populations, the number of parasite lineages increased with altitude and with the years elapsed since the establishment of sparrows in a new locality. Here, after a decline in the number of parasite lineages in the first 30 years, an increase from 40 years onwards was detected.

Main conclusions

Urbanization was related to parasite prevalence in both native and introduced bird populations. In invaded areas, altitude and time since bird introduction were related to the number of Plasmodium lineages found to be infecting sparrows.