Genetic Profiling of Plasmodium ovale wallikeri Relapses With Microsatellite Markers and Whole-Genome Sequencing.

Like Plasmodium vivax, both Plasmodium ovale curtisi and Plasmodium ovale wallikeri have the ability to cause relapse in humans, defined as recurring asexual parasitemia originating from liver-dormant forms subsequent to a primary infection. Here, we investigated relapse patterns in P ovale wallikeri infections from a cohort of travelers who were exposed to the parasite in sub-Saharan Africa and then experienced relapses after their return to France. Using a novel set of 8 highly polymorphic microsatellite markers, we genotyped 15 P ovale wallikeri relapses. For most relapses, the paired primary and relapse infections were highly genetically related (with 12 being homologous), an observation that was confirmed by whole-genome sequencing for the 4 relapses we further studied. This is, to our knowledge, the first genetic evidence of relapses in P ovale spp.

Genetic Diversity of Plasmodium falciparum and Distribution of Antimalarial Drug Resistance Mutations in Symptomatic and Asymptomatic Infections.

Malaria control relies on passive case detection, and this strategy fails detecting asymptomatic infections. In addition, infections in endemic areas harbor multiple parasite genotypes that could affect case management and malaria epidemiology. Here, we performed AmpSeq genotyping to capture polymorphisms associated with antimalarial resistance and the genetic diversity within natural Plasmodium falciparum infections. Known genetic polymorphisms associated with altered drug susceptibility were screened for the five most common marker genes, pfdhfr, pfdhps, pfmdr1, pfcrt, and pfK13, and genetic diversity was established from two known AmpSeq markers, cpmp and csp. Relative abundance of the different genotypes within mixed infections was calculated from the number of reads per genotype. Genotyping was performed on 117 samples, 63 from asymptomatic and 54 from symptomatic individuals. We identified up to 15 genotypes within an infection, and the median multiplicity of infection was higher in asymptomatic infections (median MOI = 5 in asymptomatics versus median MOI = 2 in symptomatics, P < 0.001). No genetic differentiation on parasites from asymptomatic and symptomatic individuals was found. No mutation associated with ART resistance was identified. Prevalence of the P. falciparum chloroquine resistance wild-type genotype (CVMNK) reached 80%, confirming a return to chloroquine (CQ) sensitive parasites in Cameroon. In addition, the CQ-associated resistant genotype (CVIET) was present at very low density in polyclonal infections. Persistence of low-density chloroquine resistant parasites indicates competition-survival trade-offs may contribute to maintaining genetic diversity in natura. Thus, monitoring the expansion of these low-density genotypes in different immune backgrounds will be critical to evaluate drug policy changes.

Comprehensive Analysis of Transcript and Protein Relative Abundance During Blood Stages of Plasmodium falciparum Infection.

Plasmodium falciparum is the main causative agent of human malaria. During the intraerythrocytic development cycle, the P. falciparum morphology changes dramatically from circulating young rings to sequestered mature trophozoites and schizonts. Sequestered forms contribute to the pathophysiology of severe malaria as the infected erythrocytes obstruct the microvascular flow in deep organs and induce local inflammation. However, the sequestration mechanism limits the access to the corresponding parasitic form in the clinical samples from patients infected with P. falciparum. To complement this deficiency, we aimed to evaluate the relevance of mRNA study as a proxy of protein expression in sequestered parasites. To do so, we conducted a proteotranscriptomic analysis using five independent P. falciparum laboratory strain samples. RNA sequencing was performed, and the mRNA expression level was assessed on circulating ring-stage parasites. The level of protein expression were measured by LC-MS/MS on the corresponding sequestered mature forms after 18-24 h of maturation. Overall, our results showed a strong transcriptome/transcriptome and a very strong proteome/proteome correlation between samples. Moreover, positive correlations of mRNA and protein expression levels were found between ring-stage transcriptomes and mature form proteomes. However, twice more transcripts were identified at the ring stage than proteins at the mature trophozoite stage. A high level of transcript expression did not guarantee the detection of the corresponding protein. Finally, we pointed out discrepancies at the individual gene level. Taken together, our results show that transcript and protein expressions are overall correlated. However, mRNA abundance is not a perfect proxy of protein expression at the individual level. Importantly, our study shows limitations of the "blind" use of RNA-seq and the importance of multiomics approaches for P. falciparum blood stage study in clinical samples.

Plasmodium ovale wallikeri and P. ovale curtisi Infections and Diagnostic Approaches to Imported Malaria, France, 2013-2018.

We retrospectively analyzed epidemiologic, clinical, and biologic characteristics of 368 Plasmodium ovale wallikeri and 309 P. ovale curtisi infections treated in France during January 2013­December 2018. P. ovale wallikeri infections displayed deeper thrombocytopenia and shorter latency periods. Despite similar clinical manifestations, P. ovale wallikeri­infected patients were more frequently treated with artemisinin-based combination therapy. Although the difference was not statistically significant, P. ovale wallikeri­infected patients were 5 times more frequently hospitalized in intensive care or intermediate care and had a higher proportion of severe thrombocytopenia than P. ovale curtisi­infected patients. Rapid diagnostic tests that detect aldolase were more efficient than those detecting Plasmodium lactate dehydrogenase. Sequence analysis of the potra gene from 90 P. ovale isolates reveals an insufficient polymorphism for relapse typing.

Role of miRNAs in Normal and Myasthenia Gravis Thymus.

The thymus, a primary lymphoid organ, provides a complex environment essential for the generation of the T-cell repertoire. Thymic alterations occur during life either in the context of thymic involution upon aging or the pathophysiological context of Myasthenia Gravis (MG). These changes involve complicated regulatory networks, in which microRNAs (miRNAs) are key players. Here, we analyzed the role of miRNAs in thymocyte maturation and differentiation sustained by thymic epithelial cells. We compared data from the literature regarding the role of mouse thymic miRNAs and original data obtained from a human thymic miRnome study. We identified a set of highly expressed miRNAs defined as ThymiRs and investigated miRNA expression in infants as compared to adults to determine those associated with human thymic involution. Thymic changes are also frequently observed in MG, an autoimmune disease which results in the production of anti-acetylcholine receptor (AChR) antibodies that lead to muscle weaknesses. Alterations such as thymoma in late-onset MG patients and hyperplasia with ectopic germinal centers (GCs) in early-onset (EOMG) patients are found. Thymic miRNA expression has been studied in AChR-MG patients both in thymoma-associated MG (TAMG) and EOMG, and their function through their mRNA targets investigated. Most of the dysregulated thymic miRNAs in EOMG are associated with GC development, such as miR-7, miR-24, miR-139, miR-143, miR-145, miR-146, miR-150, miR-452, miR-548 or thymic inflammation, such as miR-125b, miR-146, or miR-29. Understanding these pathways may provide therapeutic targets or biomarkers of disease manifestations.

From genomic to LC-MS/MS evidence: Analysis of PfEMP1 in Benin malaria cases.

BACKGROUND: PfEMP1 is the major protein from parasitic origin involved in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability is endless and current publicly available protein repositories do not reflect the high diversity of the sequences of PfEMP1 proteins. The identification of PfEMP1 protein sequences expressed with samples remains challenging. The aim of our study is to identify the different PfEMP1 proteins variants expressed within patient samples, and therefore identify PfEMP1 proteins domains expressed by patients presenting uncomplicated malaria or severe malaria in malaria endemic setting in Cotonou, Benin. METHODS: We performed a multi-omic approach to decipher PfEMP1 expression at the patient's level in different clinical settings. Using a combination of whole genome sequencing approach and RNA sequencing, we were able to identify new PfEMP1 sequences and created a new custom protein database. This database was used for protein identification in mass spectrometry analysis. RESULTS: The differential expression analysis of RNAsequencing data shows an increased expression of the var domains transcripts DBLα1.7, DBLα1.1, DBLα2 and DBLß12 in samples from patients suffering from Cerebral Malaria compared to Uncomplicated Malaria. Our approach allowed us to attribute PfEMP1 sequences to each sample and identify new peptides associated to PfEMP1 proteins in mass spectrometry. CONCLUSION: We highlighted the diversity of the PfEMP1 sequences from field sample compared to reference sequences repositories and confirmed the validity of our approach. These findings should contribute to further vaccine development strategies based on PfEMP1 proteins.