Blood-stage Infection Research Articles

A vaccine targeting the L9 epitope of the malaria circumsporozoite protein confers protection from blood-stage infection in a mouse challenge model

Pre-erythrocytic malaria vaccines that induce high-titer, durable antibody responses can potentially provide protection from infection. Here, we engineered a virus-like particle (VLP)-based vaccine targeting a recently described vulnerable epitope at the N-terminus of the central repeat region of the Plasmodium falciparum circumsporozoite protein that is recognized by the potently inhibitory monoclonal antibody L9 and show that immunization with L9 VLPs induces strong antibody responses that provide protection from blood-stage malaria in a mouse infection model.

Enhancing neutralization of Plasmodium falciparum using a novel monoclonal antibody against the rhoptry-associated membrane antigen

The pathogenesis of malaria is associated with blood-stage infection and there is strong evidence that antibodies specific to parasite blood-stage antigens can control parasitemia. This provides a strong rational for applying blood-stage antigen components in a multivalent vaccine, as the induced antibodies in combination can enhance protection. The Plasmodium falciparum rhoptry-associated membrane antigen (PfRAMA) is a promising vaccine target, due to its fundamental role in merozoite invasion and low level of polymorphism. Polyclonal antibodies against PfRAMA are able to inhibit P. falciparum growth and interact synergistically when combined with antibodies against P. falciparum reticulocyte-binding protein 5 (PfRh5) or cysteine-rich protective antigen (PfCyRPA). In this study, we identified a novel PfRAMA-specific mAb with neutralizing activity, which in combination with PfRh5- or PfCyRPA-specific mAbs potentiated the neutralizing effect. By applying phage display technology, we mapped the protective epitope to be in the C-terminal region of PfRAMA. Our results confirmed previous finding of synergy between PfRAMA-, PfRh5- and PfCyRPA-specific antibodies, thereby paving the way of testing these antigens (or fragments of these antigens) in combination to improve the efficacy of blood-stage malaria vaccines. The results emphasize the importance of directing antibody responses towards protective epitopes, as the majority of anti-PfRAMA mAbs were unable to inhibit merozoite invasion of erythrocytes.

Impact of chemoprophylaxis immunisation under halofantrine (CPS-HF) drug cover in Plasmodium yoelii Swiss mice malaria model.

In the present study, we have investigated the role of antimalarial drug halofantrine (HF) in inducing the sterile protection against challenges with sporozoites of the live infectious Plasmodium yoelii (Killick-Kendrick, 1967) in Swiss mice malaria model. We observed that during the first to third sequential sporozoite inoculation cycles, blood-stage patency remains the same in the control and chemoprophylaxis under HF drug cover (CPS-HF) groups. However, a delayed blood-stage infection was observed during the fourth and fifth sporozoite challenges and complete sterile protection was produced following the sixth sporozoite challenge in CPS-HF mice. We also noticed a steady decline in liver stage parasite load after 3th to 6th sporozoite challenge cycle in CPS-HF mice. CPS-HF immunisation results in a significant up-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-12 and iNOS) and down-regulation of anti-inflammatory cytokines (IL-10 and TGF-β) mRNA expression in hepatic mononuclear cells (HMNC) and spleen cells in the immunised CPS-HF mice (after 6th sporozoite challenge) compared to control. Overall, our study suggests that the repetitive sporozoite inoculation under HF drug treatment develops a strong immune response that confers protection against subsequent challenges with sporozoites of P. yoelii.

Age-specific patterns of DBLα var diversity can explain why residents of high malaria transmission areas remain susceptible to Plasmodium falciparum blood stage infection throughout life

Immunity to Plasmodium falciparum is non-sterilising, thus individuals residing in malaria-endemic areas are at risk of infection throughout their lifetime. Here we seek to find a genomic epidemiological explanation for why residents of all ages harbour blood stage infections despite lifelong exposure to P. falciparum in areas of high transmission. We do this by exploring, for the first known time, the age-specific patterns of diversity of variant antigen encoding (var) genes in the reservoir of infection. Microscopic and submicroscopic P. falciparum infections were analysed at the end of the wet and dry seasons in 2012–2013 for a cohort of 1541 residents aged from 1 to 91 years in an area characterised by high seasonal malaria transmission in Ghana. By sequencing the near ubiquitous Duffy-binding-like alpha domain (DBLα) that encodes immunogenic domains, we defined var gene diversity in an estimated 1096 genomes detected in sequential wet and dry season sampling of this cohort. Unprecedented var (DBLα) diversity was observed in all ages with 42,399 unique var types detected. There was a high degree of maintenance of types between seasons (>40% seen more than once), with many of the same types, especially upsA, appearing multiple times in isolates from different individuals. Children and adolescents were found to be significant reservoirs of var DBLα diversity compared with adults. Var repertoires within individuals were highly variable, with children having more related var repertoires compared to adolescents and adults. Individuals of all ages harboured multiple genomes with var repertoires unrelated to those infecting other hosts. High turnover of parasites with diverse isolate var repertoires was also observed in all ages. These age-specific patterns are best explained by variant-specific immune selection. The observed level of var diversity for the population was then used to simulate the development of variant-specific immunity to the diverse var types under conservative assumptions. Simulations showed that the extent of observed var diversity with limited repertoire relatedness was sufficient to explain why adolescents and adults in this community remain susceptible to blood stage infection, even with multiple genomes.

Synthesis and antiplasmodial activity of regioisomers and epimers of second-generation dual acting ivermectin hybrids

With its strong effect on vector-borne diseases, and insecticidal effect on mosquito vectors of malaria, inhibition of sporogonic and blood-stage development of Plasmodium falciparum, as well as in vitro and in vivo impairment of the P. berghei development inside hepatocytes, ivermectin (IVM) continues to represent an antimalarial therapeutic worthy of investigation. The in vitro activity of the first-generation IVM hybrids synthesized by appending the IVM macrolide with heterocyclic and organometallic antimalarial pharmacophores, against the blood-stage and liver-stage infections by Plasmodium parasites prompted us to design second-generation molecular hybrids of IVM. Here, a structural modification of IVM to produce novel molecular hybrids by using sub-structures of 4- and 8-aminoquinolines, the time-tested antiplasmodial agents used for treating the blood and hepatic stage of Plasmodium infections, respectively, is presented. Successful isolation of regioisomers and epimers has been demonstrated, and the evaluation of their in vitro antiplasmodial activity against both the blood stages of P. falciparum and the hepatic stages of P. berghei have been undertaken. These compounds displayed structure-dependent antiplasmodial activity, in the nM range, which was more potent than that of IVM, its aglycon or primaquine, highlighting the superiority of this hybridization strategy in designing new antiplasmodial agents.

Prevalence and force of Plasmodium vivax blood-stage infection and associated clinical malaria burden in the Brazilian Amazon.

BACKGROUND Understanding the epidemiology of malaria through the molecular force of the blood-stage infection of Plasmodium vivax (molFOB) may provide a detailed assessment of malaria transmission.OBJECTIVES In this study, we investigated risk factors and spatial-temporal patterns of incidence of Plasmodium infection and clinical malaria episodes in three peri-urban communities of Manaus, Western Brazilian Amazon.METHODS Monthly samples were collected in a cohort of 1,274 individuals between April 2013 and March 2014. DNA samples were subject to Plasmodium species. molFOB was calculated by counting the number of genotypes observed on each visit, which had not been present in the preceding two visits and adjusting these counts by the respective times-at-risk.FINDINGS Respectively, 77.8% and 97.2% of the population remained free of P. vivax and P. falciparum infection. Expected heterozygosity for P. vivax was 0.69 for MSP1_F3 and 0.86 for MS2. Multiplicity of infection in P. vivax was close to the value of 1. The season was associated with P. vivax positivity [adjusted hazard ratio (aHR) 2.6 (1.9-5.7)] and clinical disease [aHR 10.6 (2.4-47.2)]. P. falciparum infection was associated with previous malarial episodes [HR 9.7 (4.5-20.9)]. Subjects who reported possession of a bed net [incidence rate ratio (IRR) 1.6 (1.2-2.2)] or previous malaria episodes [IRR 3.0 (2.0-4.5)] were found to have significantly higher P. vivax molFOB.MAIN CONCLUSIONS Overall, P. vivax infection prevailed in the area and infections were mostly observed as monoclonal. Previous malaria episodes were associated with significantly higher P. vivax molFOB.

Plasmodium vivax pre-erythrocytic stages and the latent hypnozoite.

Plasmodium vivax is the most geographically widespread malaria parasite on the planet. This is largely because after mosquito transmission, P. vivax sporozoites can invade hepatocytes and form latent liver stages known as hypnozoites. These persistent liver stages can activate weeks, months or even years after an infected individual suffers a primary clinical infection. Activation then leads to replication and liver stage schizont maturation that ultimately cause relapse of blood stage infection, disease, and onward transmission. Thus, the latent hypnozoite can lie in wait during times when onward transmission is unlikely due to conditions that do not favor the mosquito. For example, in temperate climates where mosquito prevalence is only seasonal. Furthermore, the elimination of hypnozoites is challenging since the hypnozoite reservoir is currently undetectable and not killed by most antimalarial drugs. Here, we review our current knowledge of the pre-erythrocytic stages of the malaria parasite - the sporozoite and liver stages, including the elusive and enigmatic hypnozoite. We focus on our understanding of sporozoite biology, the novel animal models that are available to study the hypnozoite and hypnozoite activation and the ongoing efforts to understand the biological makeup of the hypnozoite that allow for its persistence in the human host.

Safety and efficacy of a three-dose regimen of Plasmodium falciparum sporozoite vaccine in adults during an intense malaria transmission season in Mali: a randomised, controlled phase 1 trial

WHO recently approved a partially effective vaccine that reduces clinical malaria in children, but increased vaccine activity is required to pursue malaria elimination. A phase 1 clinical trial was done in Mali, west Africa, to assess the safety, immunogenicity, and protective efficacy of a three-dose regimen of Plasmodium falciparum sporozoite (PfSPZ) Vaccine (a metabolically active, non-replicating, whole malaria sporozoite vaccine) against homologous controlled human malaria infection (CHMI) and natural P falciparum infection. We recruited healthy non-pregnant adults aged 18-50 years in Donéguébougou, Mali, and surrounding villages (Banambani, Toubana, Torodo, Sirababougou, Zorokoro) for an open-label, dose-escalation pilot study and, thereafter, a randomised, double-blind, placebo-controlled main trial. Pilot study participants were enrolled on an as-available basis to one group of CHMI infectivity controls and three staggered vaccine groups receiving: one dose of 4·5 × 105, one dose of 9 × 105, or three doses of 1·8 × 106 PfSPZ via direct venous inoculation at approximately 8 week intervals, followed by homologous CHMI 5 weeks later with infectious PfSPZ by direct venous inoculation (PfSPZ Challenge). Main cohort participants were stratified by village and randomly assigned (1:1) to receive three doses of 1·8 × 106 PfSPZ or normal saline at 1, 13, and 19 week intervals using permuted block design by the study statistician. The primary outcome was safety and tolerability of at least one vaccine dose; the secondary outcome was vaccine efficacy against homologous PfSPZ CHMI (pilot study) or against naturally transmitted P falciparum infection (main study) measured by thick blood smear. Combined artesunate and amodiaquine was administered to eliminate pre-existing parasitaemia. Outcomes were analysed by modified intention to treat (mITT; including all participants who received at least one dose of investigational product; safety and vaccine efficacy) and per protocol (vaccine efficacy). This trial is registered with ClinicalTrials.gov, number NCT02627456. Between Dec 20, 2015, and April 30, 2016, we enrolled 56 participants into the pilot study (five received the 4·5 × 105 dose, five received 9 × 105, 30 received 1·8 × 106, 15 were CHMI controls, and one withdrew before vaccination) and 120 participants into the main study cohort with 60 participants assigned PfSPZ Vaccine and 60 placebo in the main study. Adverse events and laboratory abnormalities post-vaccination in all dosing groups were few, mainly mild, and did not differ significantly between vaccine groups (all p>0·05). Unexpected severe transaminitis occured in four participants: one participant in pilot phase that received 1·8 × 106 PfSPZ Vaccine, one participant in main phase that received 1·8 × 106 PfSPZ Vaccine, and two participants in the main phase placebo group. During PfSPZ CHMI, approximately 5 weeks after the third dose of 1·8 × 106 PfSPZ, none of 29 vaccinees and one of 15 controls became positive on thick blood smear; subsequent post-hoc PCR analysis for submicroscopic blood stage infections detected P falciparum parasites in none of the 29 vaccine recipients and eight of 15 controls during CHMI. In the main trial, 32 (58%) of 55 vaccine recipients and 42 (78%) of 54 controls became positive on thick blood smear during 24-week surveillance after vaccination. Vaccine efficacy (1-hazard ratio) was 0·51 per protocol (95% CI 0·20-0·70; log-rank p=0·0042) and 0·39 by mITT (0·04-0·62; p=0·033); vaccine efficacy (1-risk ratio) was 0·24 per-protocol (0·02-0·41; p=0·031) and 0·22 mITT (0·01-0·39; p=0·041). A three-dose regimen of PfSPZ Vaccine was safe, well tolerated, and conferred 51% vaccine efficacy against intense natural P falciparum transmission, similar to 52% vaccine efficacy reported for a five-dose regimen in a previous trial. US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Sanaria. For the French translation of the abstract see Supplementary Materials section.

Recent Advances in Understanding the Inflammatory Response in Malaria: A Review of the Dual Role of Cytokines.

Malaria is a serious and, in some unfortunate cases, fatal disease caused by a parasite of the Plasmodium genus. It predominantly occurs in tropical areas where it is transmitted through the bite of an infected Anopheles mosquito. The pathogenesis of malaria is complex and incompletely elucidated. During blood-stage infection, in response to the presence of the parasite, the host's immune system produces proinflammatory cytokines including IL-6, IL-8, IFN-γ, and TNF, cytokines which play a pivotal role in controlling the growth of the parasite and its elimination. Regulatory cytokines such as transforming growth factor- (TGF-) β and IL-10 maintain the balance between the proinflammatory and anti-inflammatory responses. However, in many cases, cytokines have a double role. On the one hand, they contribute to parasitic clearance, and on the other, they are responsible for pathological changes encountered in malaria. Cytokine-modulating strategies may represent a promising modern approach in disease management. In this review, we discuss the host immune response in malaria, analyzing the latest studies on the roles of pro- and anti-inflammatory cytokines.

Chemoprophylaxis under sporozoites-lumefantrine (CPS-LMF) immunization induce protective immune responses against Plasmodium yoelii sporozoites infection in mice.

The online version contains supplementary material available at 10.1007/s13205-021-03022-0.

Type I interferon production elicits differential CD4+ T-cell responses in mice infected with Plasmodium berghei ANKA and P. chabaudi.

Plasmodium parasites that infect humans are highly polymorphic, and induce various infections ranging from an asymptomatic state to life-threatening diseases. However, how the differences between the parasites affect host immune responses during blood-stage infection remains largely unknown. We investigated the CD4+ T-cell immune responses in mice infected with P. berghei ANKA (PbA) or P. chabaudi chabaudi AS (Pcc) using PbT-II cells, which recognize a common epitope of these parasites. In the acute phase of infection, CD4+ T-cell responses in PbA-infected mice showed a lower involvement of Th1 cells and a lower proportion of Ly6Clo effector CD4+ T cells than those in Pcc-infected mice. Transcriptome analysis of PbT-II cells indicated that type I interferon (IFN)-regulated genes were expressed at higher levels in both Th1- and Tfh-type PbT-II cells from PbA-infected mice than those from Pcc-infected mice. Moreover, IFN-α levels were considerably higher in PbA-infected mice than in Pcc-infected mice. Inhibition of type I IFN signaling increased PbT-II and partially reversed the Th1 over Tfh bias of the PbT-II cells in both PbA- and Pcc-infected mice. In the memory phase, PbT-II cells in PbA-primed mice maintained higher numbers and exhibited a better recall response to the antigen. However, recall responses were not significantly different between the infection groups after re-challenge with PbA, suggesting the effect of the inflammatory environment by the infection. These observations suggest that the differences in Plasmodium-specific CD4+ T-cell responses between PbA- and Pcc-infected mice were associated with the difference in type I IFN production during the early phase of the infection.

Dramatic transcriptomic differences in Macaca mulatta and Macaca fascicularis with Plasmodium knowlesi infections

Plasmodium knowlesi, a model malaria parasite, is responsible for a significant portion of zoonotic malaria cases in Southeast Asia and must be controlled to avoid disease severity and fatalities. However, little is known about the host-parasite interactions and molecular mechanisms in play during the course of P. knowlesi malaria infections, which also may be relevant across Plasmodium species. Here we contrast P. knowlesi sporozoite-initiated infections in Macaca mulatta and Macaca fascicularis using whole blood RNA-sequencing and transcriptomic analysis. These macaque hosts are evolutionarily close, yet malaria-naïve M. mulatta will succumb to blood-stage infection without treatment, whereas malaria-naïve M. fascicularis controls parasitemia without treatment. This comparative analysis reveals transcriptomic differences as early as the liver phase of infection, in the form of signaling pathways that are activated in M. fascicularis, but not M. mulatta. Additionally, while most immune responses are initially similar during the acute stage of the blood infection, significant differences arise subsequently. The observed differences point to prolonged inflammation and anti-inflammatory effects of IL10 in M. mulatta, while M. fascicularis undergoes a transcriptional makeover towards cell proliferation, consistent with its recovery. Together, these findings suggest that timely detection of P. knowlesi in M. fascicularis, coupled with control of inflammation while initiating the replenishment of key cell populations, helps contain the infection. Overall, this study points to specific genes and pathways that could be investigated as a basis for new drug targets that support recovery from acute malaria.

Real-time PCR assays for detection and quantification of early P. falciparum gametocyte stages

The use of quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum. To understand how the parasite adjusts its transmission in response to in-host environmental conditions including antimalarials requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that specifically detect and quantify early-stage P. falciparum gametocytes. The assays are based on expression of known early and late gametocyte genes and were developed using purified stage II and stage V gametocytes and tested in natural and controlled human infections. Genes pfpeg4 and pfg27 are specifically expressed at significant levels in early gametocytes with a limit of quantification of 190 and 390 gametocytes/mL, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection. In natural infections, both early (pfpeg4/pfg27) and late gametocyte transcripts (pfs25) were detected in 71.2% of individuals, only early gametocyte transcripts in 12.6%, and only late gametocyte transcripts in 15.2%. The pfpeg4/pfg27 qRT-PCR assays are sensitive and specific for quantification of circulating sexually committed ring stages/early gametocytes and can be used to increase our understanding of epidemiological processes that modulate P. falciparum transmission.

Identification of the asymptomatic Plasmodium falciparum and Plasmodium vivax gametocyte reservoir under different transmission intensities.

BackgroundUnderstanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission.Methodology/Principal findingsPlasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44–94% for P. falciparum and from 23–72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37–100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398).Conclusions/SignificanceInterventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling.

Rodent Malaria Erythrocyte Preference Assessment by an Ex Vivo Tropism Assay.

Circulating red blood cells consist of young erythrocytes (early and late reticulocytes) and mature erythrocytes (normocytes). The human malaria parasites, Plasmodium falciparum and P. vivax, have a preference to invade reticulocytes during blood-stage infection. Rodent malaria parasites that also prefer reticulocytes could be useful tools to study human malaria reticulocyte invasion. However, previous tropism studies of rodent malaria are inconsistent from one another, making it difficult to compare cell preference of different parasite species and strains. In vivo measurements of cell tropism are also subjected to many confounding factors. Here we developed an ex vivo tropism assay for rodent malaria with highly purified fractions of murine reticulocytes and normocytes. We measured invasion into the different erythrocyte populations using flow cytometry and evaluated the tropism index of the parasite strains. We found that P. berghei ANKA displayed the strongest reticulocyte preference, followed by P. yoelii 17X1.1, whereas P. chabaudi AS and P. vinckei S67 showed mixed tropism. These preferences are intrinsic and were maintained at different reticulocyte and normocyte availabilities. Our study shed light on the true erythrocyte preference of the parasites and paves the way for future investigations on the receptor-ligand interactions mediating erythrocyte tropism.

Screening of viral-vectored P. falciparum pre-erythrocytic candidate vaccine antigens using chimeric rodent parasites.

To screen for additional vaccine candidate antigens of Plasmodium pre-erythrocytic stages, fourteen P. falciparum proteins were selected based on expression in sporozoites or their role in establishment of hepatocyte infection. For preclinical evaluation of immunogenicity of these proteins in mice, chimeric P. berghei sporozoites were created that express the P. falciparum proteins in sporozoites as an additional copy gene under control of the uis4 gene promoter. All fourteen chimeric parasites produced sporozoites but sporozoites of eight lines failed to establish a liver infection, indicating a negative impact of these P. falciparum proteins on sporozoite infectivity. Immunogenicity of the other six proteins (SPELD, ETRAMP10.3, SIAP2, SPATR, HT, RPL3) was analyzed by immunization of inbred BALB/c and outbred CD-1 mice with viral-vectored (ChAd63 or ChAdOx1, MVA) vaccines, followed by challenge with chimeric sporozoites. Protective immunogenicity was determined by analyzing parasite liver load and prepatent period of blood stage infection after challenge. Of the six proteins only SPELD immunized mice showed partial protection. We discuss both the low protective immunogenicity of these proteins in the chimeric rodent malaria challenge model and the negative effect on P. berghei sporozoite infectivity of several P. falciparum proteins expressed in the chimeric sporozoites.

Mechanistic within-host models of the asexual Plasmodium falciparum infection: a review and analytical assessment

BackgroundMalaria blood-stage infection length and intensity are important drivers of disease and transmission; however, the underlying mechanisms of parasite growth and the host’s immune response during infection remain largely unknown. Over the last 30 years, several mechanistic mathematical models of malaria parasite within-host dynamics have been published and used in malaria transmission models.MethodsMechanistic within-host models of parasite dynamics were identified through a review of published literature. For a subset of these, model code was reproduced and descriptive statistics compared between the models using fitted data. Through simulation and model analysis, key features of the models were compared, including assumptions on growth, immune response components, variant switching mechanisms, and inter-individual variability.ResultsThe assessed within-host malaria models generally replicate infection dynamics in malaria-naïve individuals. However, there are substantial differences between the model dynamics after disease onset, and models do not always reproduce late infection parasitaemia data used for calibration of the within host infections. Models have attempted to capture the considerable variability in parasite dynamics between individuals by including stochastic parasite multiplication rates; variant switching dynamics leading to immune escape; variable effects of the host immune responses; or via probabilistic events. For models that capture realistic length of infections, model representations of innate immunity explain early peaks in infection density that cause clinical symptoms, and model representations of antibody immune responses control the length of infection. Models differed in their assumptions concerning variant switching dynamics, reflecting uncertainty in the underlying mechanisms of variant switching revealed by recent clinical data during early infection. Overall, given the scarce availability of the biological evidence there is limited support for complex models.ConclusionsThis study suggests that much of the inter-individual variability observed in clinical malaria infections has traditionally been attributed in models to random variability, rather than mechanistic disease dynamics. Thus, it is proposed that newly developed models should assume simple immune dynamics that minimally capture mechanistic understandings and avoid over-parameterization and large stochasticity which inaccurately represent unknown disease mechanisms.

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells

BackgroundCultured human red blood cells (RBCs) provide a powerful ex vivo assay platform to study blood-stage malaria infection and propagation. In recent years, high-resolution metabolomic methods have quantified hundreds of metabolites from parasite-infected RBC cultures under a variety of perturbations. In this context, the corresponding control samples of the uninfected culture systems can also be used to examine the effects of these perturbations on RBC metabolism itself and their dependence on blood donors (inter-study variations).MethodsTime-course datasets from five independent studies were generated and analysed, maintaining uninfected RBCs (uRBC) at 2% haematocrit for 48 h under conditions originally designed for parasite cultures. Using identical experimental protocols, quadruplicate samples were collected at six time points, and global metabolomics were employed on the pellet fraction of the uRBC cultures. In total, ~ 500 metabolites were examined across each dataset to quantify inter-study variability in RBC metabolism, and metabolic network modelling augmented the analyses to characterize the metabolic state and fluxes of the RBCs.ResultsTo minimize inter-study variations unrelated to RBC metabolism, an internal standard metabolite (phosphatidylethanolamine C18:0/20:4) was identified with minimal variation in abundance over time and across all the samples of each dataset to normalize the data. Although the bulk of the normalized data showed a high degree of inter-study consistency, changes and variations in metabolite levels from individual donors were noted. Thus, a total of 24 metabolites were associated with significant variation in the 48-h culture time window, with the largest variations involving metabolites in glycolysis and synthesis of glutathione. Metabolic network analysis was used to identify the production of superoxide radicals in cultured RBCs as countered by the activity of glutathione oxidoreductase and synthesis of reducing equivalents via the pentose phosphate pathway. Peptide degradation occurred at a rate that is comparable with central carbon fluxes, consistent with active degradation of methaemoglobin, processes also commonly associated with storage lesions in RBCs.ConclusionsThe bulk of the data showed high inter-study consistency. The collected data, quantification of an expected abundance variation of RBC metabolites, and characterization of a subset of highly variable metabolites in the RBCs will help in identifying non-specific changes in metabolic abundances that may obscure accurate metabolomic profiling of Plasmodium falciparum and other blood-borne pathogens.

Plasmodium vivax Latent Liver Stage Infection and Relapse: Biological Insights and New Experimental Tools.

Plasmodium vivax is the most widespread human malaria parasite, in part because it can form latent liver stages known as hypnozoites after transmission by female anopheline mosquitoes to human hosts. These persistent stages can activate weeks, months, or even years after the primary clinical infection; replicate; and initiate relapses of blood stage infection, which causes disease and recurring transmission. Eliminating hypnozoites is a substantial obstacle for malaria treatment and eradication since the hypnozoite reservoir is undetectable and unaffected by most antimalarial drugs. Importantly, in some parts of the globe where P. vivax malaria is endemic, as many as 90% of P. vivax blood stage infections are thought to be relapses rather than primary infections, rendering the hypnozoite a major driver of P. vivax epidemiology. Here, we review the biology of the hypnozoite and recent discoveries concerning this enigmatic parasite stage. We discuss treatment and prevention challenges, novel animal models to study hypnozoites and relapse, and hypotheses related to hypnozoite formation and activation.

Loop-mediated isothermal amplification (LAMP) assays targeting 18S ribosomal RNA genes for identifying P. vivax and P. ovale species and mitochondrial DNA for detecting the genus Plasmodium

BackgroundLoop-mediated isothermal amplification (LAMP) has been widely used to diagnose various infectious diseases. Malaria is a globally distributed infectious disease attributed to parasites in the genus Plasmodium. It is known that persons infected with Plasmodium vivax and P. ovale are prone to clinical relapse of symptomatic blood-stage infections. LAMP has not previously been specifically evaluated for its diagnostic performance in detecting P. ovale in an epidemiological study, and no commercial LAMP or rapid diagnostic test (RDT) kits are available for specifically diagnosing infections with P. ovale.MethodsAn assay was designed to target a portion of mitochondrial DNA (mtDNA) among Plasmodium spp., the five human Plasmodium species and two other assays were designed to target the nuclear 18S ribosomal DNA gene (18S rDNA) of either P. vivax or P. ovale for differentiating the two species. The sensitivity of the assays was compared to that of nested PCR using defined concentrations of plasmids containing the target sequences and using limiting dilutions prepared from clinical isolates derived from Chinese workers who had become infected in Africa or near the Chinese border with Myanmar.ResultsThe results showed that 102 copies of the mitochondrial target or 102 and 103 copies of 18S rDNA could be detected from Plasmodium spp., P. vivax and P. ovale, respectively. In 279 clinical samples, the malaria Pan mtDNA LAMP test performed well when compared with a nested PCR assay (95% confidence interval [CI] sensitivity 98.48–100%; specificity 90.75–100%). When diagnosing clinical cases of infection with P. vivax, the 18S rDNA assay demonstrated an even great sensitivity (95.85–100%) and specificity (98.1–100%). The same was true for clinical infections with P. ovale (sensitivity 90.76–99.96%; specificity 98.34–100%). Using plasmid-positive controls, the limits of detection of Malaria Pan, 18S rDNA P. vivax and 18S rDNA P. ovale LAMP were 100-, 100- and tenfold lower than those of PCR, respectively.ConclusionThe novel LAMP assays can greatly aid the rapid, reliable and highly sensitive diagnosis of infections of Plasmodium spp. transmitted among people, including P. vivax and P. ovale, cases of which are most prone to clinical relapse.Graphical