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Background Artemisinin-based combination therapies (ACTs) are the first-line treatments for uncomplicated malaria in Ghana. Artemisinin (ART) tolerance in Plasmodium falciparum has arisen in Southeast Asia (SEA) and recently, in parts of East Africa. This is ascribed to the survival of ring-stage parasites post treatment. We sought to assess and characterize correlates of potential ART tolerance based on post-treatment parasite clearance, ex vivo and in vitro drug vulnerability, and molecular markers of drug resistance in P. falciparum isolates from children with uncomplicated malaria in Ghana. Methods Six months to fourteen years old children having uncomplicated malaria (n = 115) were enrolled from two hospitals and a health center in Ghana’s Greater Accra region and treated with artemether-lumefantrine (AL) according to body weight. Pre- and post-treatment parasitemia (day 0 and day 3) was confirmed by microscopy. The ex vivo ring-stage survival assay (RSA) was used to detect percent ring survival while the 72 hr SYBR Green 1 assay was used to measure the 50% inhibition concentration (IC50s) of ART and its derivatives and partner drugs. Genetic markers of drug tolerance /resistance were evaluated using selective whole genome sequencing. Results Of the total of 115 participants, 85 were successfully followed up on day 3 post-treatment and had 2/85 (2.4%) parasitemia. The IC50 values of ART, artesunate (AS), artemether (AM), dihydroartemisinin (DHA), amodiaquine (AQ), and lumefantrine (LUM) were not indicative of drug tolerance. However, 7/90 (7.8%) pre-treatment isolates had > 10% ring survival rates against DHA. Of the four isolates (2 RSA positive and 2 RSA negative) with high genomic coverage, P. falciparum kelch 13 K188* and Pfcoronin V424I mutations were only present in the two RSA positive isolates with > 10% ring survival rates. Conclusions The observed low proportion of participants with post-treatment parasitemia suggests AL remains efficacious. However, the increased rates of survival observed in the ex vivo RSA against DHA, maybe a pointer of an early start of ART tolerance. Furthermore, the role of two novel mutations in PfK13 and Pfcoronin genes, harbored by the two RSA positive isolates that had high ring survival in our study, remains to be elucidated.

Sequestration of Plasmodium falciparum(P. falciparum)-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins is central to the development of malaria pathogenesis. PfEMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum-infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, PfEMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var-expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with PfEMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding PfEMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic PfEMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites.

SummaryAssessing the diversity or expression of variable gene families in pathogens can inform about immune escape mechanisms or host interaction phenotypes of clinical relevance. However, obtaining the sequences and quantifying their expression is a challenge. Here, we present a tool, which based on unique sequence tag similarity between members of a gene family, predicts the domains encoded by the queried gene. As an example, we are using the var gene family, encoding the major virulence proteins (PfEMP1) of the human malaria parasite, Plasmodium falciparum. We developed Varia, which predicts the likely var gene sequence and encoded protein domain composition of a gene from short sequence tags. We provide a new extended annotated var genome database, in which Varia identifies genes with identical tag sequences and compares these to return the most probable domain composition of the query gene. Varia’s ability to predict correct PfEMP1 domain compositions from short var sequence tags was tested in two complementary pipelines to (a) return the putative gene sequences and domain compositions of the query gene from any partial sequence provided, thereby enabling detailed assessment of specific genes’ putative function and experimental validation of these (b) to accommodate rapid profiling of var gene expression in complex patient samples, by compiling the overall domain prevalence among var transcripts predicted identified and quantified by next generation sequencing of so-called var DBLα-sequence tags.Availability and implementationVaria is available on GitHub (https://github.com/GCJMacken-zie/Varia) under the MIT license.Contactthomasl@sund.ku.dk, thomasdan.otto@glasgow.ac.uk

Mutations in the Plasmodium falciparum genes Pfdhfr and Pfdhps, particularly the sextuple mutant haplotype threatens the antimalarial effectiveness of sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment during pregnancy (IPTp). To explore the impact of sextuple mutant haplotype infections on outcome measures after provision of IPTp with SP, we monitored birth outcomes in women followed up from before conception or from the first trimester until delivery. Women infected with sextuple haplotypes, in the early second trimester specifically, delivered newborns with a lower birth weight compared with women who did not have malaria during pregnancy (difference, −267 g; 95% confidence interval, −454 to −59; P = .01) and women infected with less SP-resistant haplotypes (−461 g; −877 to −44; P = .03). Thus, sextuple haplotype infections seem to affect the effectiveness of SP for IPTp and directly affect birth outcome by lowering birth weight. Close monitoring and targeted malaria control during early pregnancy is therefore crucial to improving birth outcomes.