Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples' features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.

Transcriptional profiling of hepatocytes infected with the replicative form of the malaria parasite Plasmodium cynomolgi.

BACKGROUND: The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. The transcriptional profiling of P. cynomolgi liver stages was previously reported and revealed many important biological features of the parasite but left out the host response to malaria infection. METHODS: Previously published RNA sequencing data were used to quantify the expression of host genes in rhesus macaque hepatocytes infected with P. cynomolgi in comparison to either cells from uninfected samples or uninfected bystander cells. RESULTS: Although the dataset could not be used to resolve the transcriptional profile of hypnozoite-infected hepatocytes, it provided a snapshot of the host response to liver stage schizonts at 9-10 day post-infection and identified specific host pathways that are modulated during the exo-erythrocytic stage of P. cynomolgi. CONCLUSIONS: This study constitutes a valuable resource characterizing the hepatocyte response to P. cynomolgi infection and provides a framework to build on future research that aims at understanding hepatocyte-parasite interactions during relapsing malaria infection.

Population-level estimates of the proportion of Plasmodium vivax blood-stage infections attributable to relapses among febrile patients attending Adama Malaria Diagnostic Centre, East Shoa Zone, Oromia, Ethiopia.

BACKGROUND: Malaria is ranked as the leading communicable disease in Ethiopia, where Plasmodium falciparum and Plasmodium vivax are co-endemic. The incidence of P. vivax is usually considered to be less seasonal than P. falciparum. Clinical cases of symptomatic P. falciparum exhibit notable seasonal variation, driven by rainfall-dependent variation in the abundance of Anopheles mosquitoes. A similar peak of clinical cases of P. vivax is usually observed during the rainy season. However, the ability of P. vivax to relapse causing new blood-stage infections weeks to months after an infectious mosquito bite can lead to substantial differences in seasonal patterns of clinical cases. These cannot be detected with currently available diagnostic tools and are not cleared upon treatment with routinely administered anti-malarial drugs. METHODS: A health- facility based cross-sectional study was conducted in Adama malaria diagnostic centre from May 2015 to April 2016. Finger-prick blood samples were collected for thin and thick blood film preparation from participants seeking treatment for suspected cases of febrile malaria. Informed consent was obtained from each study participant or their guardians. Seasonal patterns in malaria cases were analysed using statistical models, identifying the peaks in cases, and the seasonally varying proportion of P. vivax cases attributable to relapses. RESULTS: The proportion of patients with malaria detectable by light microscopy was 36.1% (1141/3161) of which P. vivax, P. falciparum, and mixed infections accounted for 71.4, 25.8 and 2.8%, respectively. Of the febrile patients diagnosed, 2134 (67.5%) were males and 1919 (60.7%) were urban residents. The model identified a primary peak in P. falciparum and P. vivax cases from August to October, as well as a secondary peak of P. vivax cases from February to April attributable to cases arising from relapses. During the secondary peak of P. vivax cases approximately 77% (95% CrI 68, 84%) of cases are estimated to be attributable to relapses. During the primary peak from August to October, approximately 40% (95% CrI 29, 57%) of cases are estimated to be attributable to relapses. DISCUSSION: It is not possible to diagnose whether a P. vivax case has been caused by blood-stage infection from a mosquito bite or a relapse. However, differences in seasonal patterns of P. falciparum and P. vivax cases can be used to estimate the population-level proportion of P. vivax cases attributable to relapses. These observations have important implications for the epidemiological assessment of vivax malaria, and initiating therapy that is effective against both blood stages and relapses.

Clinical implications of a gradual dormancy concept in malaria.

Malaria recurrences after an initially successful therapy and malarial fever occurring a long time after infection are well-known problems in malariology. Currently, two distinct types of malaria recurrences are defined: recrudescence and relapse. A recrudescence is thought to originate from circulating Plasmodium blood stages which do not cause fever before a certain level of a microscopically detectable parasitemia is reached. Contrary, a relapse is thought to originate from quiescent intracellular hepatic parasite stages called hypnozoites. Recrudescences would typically occur in infections due to Plasmodium falciparum. Plasmodium knowlesi, and Plasmodium malariae, whereas relapses would be caused exclusively by Plasmodium vivax and Plasmodium ovale. This schematic view is, however, insufficiently supported by experimental evidence. For instance, hypnozoites of P. ovale have never been experimentally documented. On the other hand, the nonfinding of P. malariae hypnozoites turned into the proof for the nonexistence of P. malariae hypnozoites. Clinical relapse-type recurrences have been observed in both P. ovale and P. malariae infections, and decade-long incubation times have also been reported in P. falciparum infections. We propose a gradual hypothesis in accordance with the continuity concept of biological evolution: both, relapse and recrudescence may be potentially caused by all Plasmodium spp. We hypothesize that the difference between the various Plasmodium spp. is quantitative rather than qualitative: there are Plasmodium spp. which frequently cause relapses such as P. vivax, particularly the P.v. Chesson strain, species which cause relapses less frequently, such as P. ovale and sometimes P. malariae, and species which may exceptionally cause relapses such as P. falciparum. All species may cause recrudescences. As clinical consequences, we propose that 8-aminquinolines may be considered in a relapse-type recrudescence regardless of the causal Plasmodium sp., whereas primaquine relapse prevention might not be routinely indicated in malaria due to P. ovale.

Vivax malaria: a possible stumbling block for malaria elimination in India.

Plasmodium vivax is geographically the most widely dispersed human malaria parasite species. It has shown resilience and a great deal of adaptability. Genomic studies suggest that P. vivax originated from Asia or Africa and moved to the rest of the world. Although P. vivax is evolutionarily an older species than Plasmodium falciparum, its biology, transmission, pathology, and control still require better elucidation. P. vivax poses problems for malaria elimination because of the ability of a single primary infection to produce multiple relapses over months and years. P. vivax malaria elimination program needs early diagnosis, and prompt and complete radical treatment, which is challenging, to simultaneously exterminate the circulating parasites and dormant hypnozoites lodged in the hepatocytes of the host liver. As prompt surveillance and effective treatments are rolled out, preventing primaquine toxicity in the patients having glucose-6-phosphate dehydrogenase (G6PD) deficiency should be a priority for the vivax elimination program. This review sheds light on the burden of P. vivax, changing epidemiological patterns, the hurdles in elimination efforts, and the essential tools needed not just in India but globally. These tools encompass innovative treatments for eliminating dormant parasites, coping with evolving drug resistance, and the development of potential vaccines against the parasite.

Plasmodium vivax - How hidden reservoirs hinder global malaria elimination.

Plasmodium vivax is the most geographically widespread human malaria parasite. Global malaria efforts have been less successful at reducing the burden of P. vivax compared to P. falciparum, owing to the unique biology and related treatment complexity of P. vivax. As a result, P. vivax is now the dominant malaria parasite throughout the Asia-Pacific and South America causing up to 14 million clinical cases every year and is considered a major obstacle to malaria elimination. Key features circumventing existing malaria control tools are the transmissibility of asymptomatic, low-density circulating infections and reservoirs of persistent dormant liver stages (hypnozoites) that are undetectable but reactivate to cause relapsing infections and sustain transmission. In this review we summarise the new knowledge shaping our understanding of the global epidemiology of P. vivax infections, highlighting the challenges for elimination and the tools that will be required achieve this.

A single-cell liver atlas of Plasmodium vivax infection.

Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years and reactivate to cause recurrent blood-stage infection. Although they are an important target for malaria eradication, little is known about the molecular features of replicative and non-replicative intracellular liver-stage parasites and their host cell dependence. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites for transcriptional profiling. Coupling enrichment strategies with bulk and single-cell analyses, we capture both parasite and host transcripts in individual hepatocytes throughout the course of infection. We define host- and state-dependent transcriptional signatures and identify unappreciated populations of replicative and non-replicative parasites that share features with sexual transmissive forms. We find that infection suppresses the transcription of key hepatocyte function genes and elicits an anti-parasite innate immune response. Our work provides a foundation for understanding host-parasite interactions and reveals insights into the biology of P. vivax dormancy and transmission.

Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Anti-relapse Compounds.

Malaria hypnozoites are dormant parasite stages that reside inside hepatocytes. Upon activation, these stages can resume growth, causing new episodes of blood stage malaria infection. This chapter describes a fast and sensitive protocol for the detection of bioluminescent (BL) hypnozoites in vitro. Using transgenic Plasmodium cynomolgi parasites that differentially express the BL reporter proteins firefly luciferase and the ultrabright NanoLuc, hypnozoites can be distinguished from liver stage schizonts. This robust method sets the stage for implementation in large-scale drug screening platforms with the aim to find new compounds that eliminate hypnozoites.

Evolutionary genetics of malaria.

Many standard-textbook population-genetic results apply to a wide range of species. Sometimes, however, population-genetic models and principles need to be tailored to a particular species. This is particularly true for malaria, which next to tuberculosis and HIV/AIDS ranks among the economically most relevant infectious diseases. Importantly, malaria is not one disease-five human-pathogenic species of Plasmodium exist. P. falciparum is not only the most severe form of human malaria, but it also causes the majority of infections. The second most relevant species, P. vivax, is already considered a neglected disease in several endemic areas. All human-pathogenic species have distinct characteristics that are not only crucial for control and eradication efforts, but also for the population-genetics of the disease. This is particularly true in the context of selection. Namely, fitness is determined by so-called fitness components, which are determined by the parasites live-history, which differs between malaria species. The presence of hypnozoites, i.e., dormant liver-stage parasites, which can cause disease relapses, is a distinct feature of P. vivax and P. ovale sp. In P. malariae inactivated blood-stage parasites can cause a recrudescence years after the infection was clinically cured. To properly describe population-genetic processes, such as the spread of anti-malarial drug resistance, these features must be accounted for appropriately. Here, we introduce and extend a population-genetic framework for the evolutionary dynamics of malaria, which applies to all human-pathogenic malaria species. The model focuses on, but is not limited to, the spread of drug resistance. The framework elucidates how the presence of dormant liver stage or inactivated blood stage parasites that act like seed banks delay evolutionary processes. It is shown that, contrary to standard population-genetic theory, the process of selection and recombination cannot be decoupled in malaria. Furthermore, we discuss the connection between haplotype frequencies, haplotype prevalence, transmission dynamics, and relapses or recrudescence in malaria.