A conserved Plasmodium nuclear protein is critical for late liver stage development.

Malaria, caused by Plasmodium parasites, imposes a significant health burden and live-attenuated parasites are being pursued as vaccines. Here, we report on the creation of a genetically attenuated parasite by the deletion of Plasmodium LINUP, encoding a liver stage nuclear protein. In the rodent parasite Plasmodium yoelii, LINUP expression was restricted to liver stage nuclei after the onset of liver stage schizogony.

Assessing the daily natural history of asymptomatic Plasmodium infections in adults and older children in Katakwi, Uganda: a longitudinal cohort study.

Background: Low-density asymptomatic Plasmodium infections are prevalent in endemic areas, but little is known about their natural history. The trajectories of these infections and their propensity to fluctuate to undetectable densities can affect detection in clinical trials and field studies. We aimed to classify the natural history of these infections in a high transmission area over 29 days.

More time to kill: A longer liver stage increases T cell-mediated protection against pre-erythrocytic malaria.

Liver stage (LS) Plasmodia mature in 2-2.5 days in rodents compared to 5-6 days in humans. -specific CD8 T cell expansion differs across these varied timespans.

Plasmodium knowlesi in pig-tailed macaques: a potential new model for malaria vaccine research.

Background: Plasmodium knowlesi is an established experimental model for basic and pre-clinical malaria vaccine research. Historically, rhesus macaques have been the most common host for malaria vaccine studies with P. knowlesi parasites.

18S Ribosomal RNA Biomarker Clearance After Food and Drug Administration-Approved Antimalarial Treatment in Controlled Human Malaria Infection Trials.

Background: Sensitive molecular assays, such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of 18S ribosomal RNA (rRNA), are increasingly the primary method of detecting infections in controlled human malaria infection (CHMI) trials. However, thick blood smears (TBSs) remain the main method for confirming clearance of parasites after curative treatment, in part owing to uncertainty regarding biomarker clearance rates.

Methods: For this analysis, 18S rRNA qRT-PCR data were compiled from 127 -infected participants treated with chloroquine or atovaquone-proguanil in 6 CHMI studies conducted in Seattle, Washington, over the past decade.

Rethinking detection of pre-existing and intervening infections in malaria clinical trials.

Pre-existing and intervening low-density infections complicate the conduct of malaria clinical trials. These infections confound infection detection endpoints, and their immunological effects may detract from intended vaccine-induced immune responses. Historically, these infections were often unrecognized since infrequent and often analytically insensitive parasitological testing was performed before and during trials.

Needle-free, spirulina-produced Plasmodium falciparum circumsporozoite vaccination provides sterile protection against pre-erythrocytic malaria in mice.

Antibodies against the Plasmodium falciparum circumsporozoite protein (PfCSP) can block hepatocyte infection by sporozoites and protect against malaria. Needle-free vaccination strategies are desirable, yet most PfCSP-targeted vaccines like RTS,S require needle-based administration. Here, we evaluated the edible algae, Arthrospira platensis (commonly called 'spirulina') as a malaria vaccine platform.

A genetically engineered parasite vaccine provides protection from controlled human malaria infection.

Genetically engineered live sporozoites constitute a potential platform for creating consistently attenuated, genetically defined, whole-parasite vaccines against malaria through targeted gene deletions. Such genetically attenuated parasites (GAPs) do not require attenuation by irradiation or concomitant drug treatment. We previously developed a (Pf) GAP with deletions in , , and genes (PfGAP3KO) and demonstrated its safety and immunogenicity in humans.

Feasibility of community at-home dried blood spot collection combined with pooled reverse transcription PCR as a viable and convenient method for malaria epidemiology studies.

Background: Many Plasmodium infections in endemic regions exist at densities below the limit of detection of standard diagnostic tools. These infections threaten control efforts and may impact vaccine and therapeutic drug studies. Simple, cost-effective methods are needed to study the natural history of asymptomatic submicroscopic parasitaemia.

Anti-TRAP/SSP2 monoclonal antibodies can inhibit sporozoite infection and may enhance protection of anti-CSP monoclonal antibodies.

Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP).

Flow Cytometric Sorting of Infected Erythrocytes Demonstrates Reliable Detection of Individual Ring-Stage Plasmodium falciparum Parasites by Plasmodium 18S rRNA Reverse Transcription Polymerase Chain Reaction.

Molecular diagnostic tests for Plasmodium falciparum parasites are increasingly used to enable ultrasensitive detection of infection in clinical trials and field surveillance studies. Ribonucleic acid (RNA)-based assays targeting 18S rRNA are particularly sensitive with limits of detection reported to comprise a single infected red blood cell (RBC) in a relatively large volume of blood. However, the validation testing at such limiting concentrations is hampered by the so-called Poisson distribution of such rare events, which can lead laboratorians to inaccurately set the limit of detection higher (i.

Cryopreserved Sporozoites with and without the Glycolipid Adjuvant 7DW8-5 Protect in Prime-and-Trap Malaria Vaccination.

Repeated intravenous (IV) administration of radiation-attenuated sporozoite (RAS) vaccines induces Plasmodium-specific CD8+ liver-resident memory T (Trm) cells in mice and achieves sterile protection against challenge. Our heterologous "prime-and-trap" vaccine strategy was previously shown to simplify and improve upon RAS vaccination. Prime-and-trap vaccination combines epidermal priming by DNA-encoded circumsporozoite protein (CSP) antigen followed by a single IV dose of freshly dissected RAS (fresh-RAS) to direct and trap activated and expanding CD8+ T cells in the liver.

Application of dried blood spot sample pooling strategies for Plasmodium 18S rRNA biomarker testing to facilitate identification of infected persons in large-scale epidemiological studies.

Background: Plasmodium 18S rRNA is a sensitive biomarker for detecting Plasmodium infection in human blood. Dried blood spots (DBS) are a practical sample type for malaria field studies to collect, store, and transport large quantities of blood samples for diagnostic testing. Pooled testing is a common way to reduce reagent costs and labour.

Clustering of subpatent infections in households with asymptomatic rapid diagnostic test-positive cases in Bioko Island, Equatorial Guinea independent of travel to regions of higher malaria endemicity: a cross-sectional study.

Background: Prevalence of falciparum malaria on Bioko Island remains high despite sustained, intensive control. Progress may be hindered by high proportions of subpatent infections that are not detected by rapid diagnostic tests (RDT) but contribute to onward transmission, and by imported infections. Better understanding of the relationship between subpatent infections and RDT-detected infections, and whether this relationship is different from imported versus locally acquired infections, is imperative to better understand the sources of infection and mechanisms of transmission to tailor more effective interventions.

Quantification of Plasmodium knowlesi versus Plasmodium falciparum in the rhesus liver: implications for malaria vaccine studies in rhesus models.

Background: Rhesus macaques are valuable pre-clinical models for malaria vaccine development. The Plasmodium knowlesi/rhesus and Plasmodium falciparum/rhesus models are two established platforms for malaria vaccine testing, and both have previously been used to assess live-attenuated sporozoite vaccines. However, there is evidence that the susceptibility of the rhesus liver to P.

Safety, Pharmacokinetics, and Causal Prophylactic Efficacy of KAF156 in a Plasmodium falciparum Human Infection Study.

Background: KAF156 is a novel antimalarial drug that is active against both liver- and blood-stage Plasmodium parasites, including drug-resistant strains. Here, we investigated the causal prophylactic efficacy of KAF156 in a controlled human malaria infection (CHMI) model.

Methods: In part 1, healthy, malaria-naive participants received 800 mg KAF156 or placebo 3 hours before CHMI with P.

Beyond Blood Smears: Qualification of 18S rRNA as a Biomarker for Controlled Human Malaria Infections.

18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy.

Malaria Parasite Density in Individuals with Different Rapid Diagnostic Test Results and Concentrations of HRP2 Antigen.

Low-density malaria infections are a source of human morbidity in endemic settings and potentially contribute to ongoing malaria transmission. Conventional rapid diagnostic tests (RDTs) were designed to detect clinically relevant parasite and antigen levels, but it is largely unknown what proportion of parasite (and antigen positive) infections are missed by conventional RDTs. Furthermore, RDTs can also provide false positives from lingering histidine-rich protein 2 (HRP2) antigenemia from a past infection.

Simultaneous Quantification of Antigens and Host Factor C-Reactive Protein in Asymptomatic Individuals with Confirmed Malaria by Use of a Novel Multiplex Immunoassay.

Malaria rapid diagnostic tests (RDTs) primarily detect antigen histidine-rich protein 2 (HRP2) and the malaria-conserved antigen lactate dehydrogenase (LDH) for and other malaria species. The performance of RDTs and their utility is dependent on circulating antigen concentration distributions in infected individuals in a population in which malaria is endemic and on the limit of detection of the RDT for the antigens. A multiplexed immunoassay for the quantification of HRP2, LDH, and all-malaria LDH (pan LDH) was developed to accurately measure circulating antigen concentration and antigen distribution in a population with endemic malaria.

Screening for Pfhrp2/3-Deleted Plasmodium falciparum, Non-falciparum, and Low-Density Malaria Infections by a Multiplex Antigen Assay.

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis.

Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) antigens.

Plasmodium 18S rRNA of intravenously administered sporozoites does not persist in peripheral blood.

Background: Plasmodium 18S rRNA is a biomarker used to monitor blood-stage infections in malaria clinical trials. Plasmodium sporozoites also express this biomarker, and there is conflicting evidence about how long sporozoite-derived 18S rRNA persists in peripheral blood. If present in blood for an extended timeframe, sporozoite-derived 18S rRNA could complicate use as a blood-stage biomarker.

Diagnostic challenges of prolonged post-treatment clearance of Plasmodium nucleic acids in a pre-transplant autosplenectomized patient with sickle cell disease.

Background: Autosplenectomy, as a result of sickle cell disease, is an important risk factor for severe malaria. While molecular methods are helpful in providing rapid and accurate infection detection and species identification, the effect of hyposplenism on result interpretation during the course of infection should be carefully considered.

Case Presentation: A 32-year old autosplenectomized Nigerian male with severe sickle cell disease was referred to the National Institutes of Health for allogenic hematopoietic stem cell transplant.

Performance of a High-Sensitivity Rapid Diagnostic Test for Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections.

Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.