PHARE: a bioinformatics pipeline for compositional profiling of multiclonal Plasmodium falciparum infections from long-read Nanopore sequencing data.

BACKGROUND: The emergence of drug-resistant clones of Plasmodium falciparum is a major public health concern, and the ability to detect and track the spread of these clones is crucial for effective malaria control and treatment. However, in endemic settings, malaria infected people often carry multiple P. falciparum clones simultaneously making it likely to miss drug-resistant clones using traditional molecular typing methods. OBJECTIVES: Our goal was to develop a bioinformatics pipeline for compositional profiling in multiclonal P. falciparum samples, sequenced using the Oxford Nanopore Technologies MinION platform. METHODS: We developed the 'Finding P. falciparum haplotypes with resistance mutations in polyclonal infections' (PHARE) pipeline using existing bioinformatics tools and custom scripts written in python. PHARE was validated on three control datasets containing P. falciparum DNA of four laboratory strains at varying mixing ratios. Additionally, the pipeline was tested on clinical samples from children admitted to a paediatric hospital in the Central African Republic. RESULTS: The PHARE pipeline achieved high recall and accuracy rates in all control datasets. The pipeline can be used on any gene and was tested with amplicons of the P. falciparum drug resistance marker genes pfdhps, pfdhfr and pfK13. CONCLUSIONS: The PHARE pipeline helps to provide a more complete picture of drug resistance in the circulating P. falciparum population and can help to guide treatment recommendations. PHARE is freely available under the GNU Lesser General Public License v.3.0 on GitHub: https://github.com/Fippu/PHARE.

Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019).

BACKGROUND: Over the last decade, artemisinin-based combination therapy (ACT) has contributed substantially to the decrease in malaria-related morbidity and mortality. The emergence of Plasmodium falciparum parasites resistant to artemisinin derivatives in Southeast Asia and the risk of their spread or of local emergence in sub-Saharan Africa are a major threat to public health. This study thus set out to estimate the proportion of P. falciparum isolates, with Pfkelch13 gene mutations associated with artemisinin resistance previously detected in Southeast Asia. METHODS: Blood samples were collected in two sites of Bangui, the capital of the Central African Republic (CAR) from 2017 to 2019. DNA was extracted and nested PCR were carried out to detect Plasmodium species and mutations in the propeller domain of the Pfkelch13 gene for P. falciparum samples. RESULTS: A total of 255 P. falciparum samples were analysed. Plasmodium ovale DNA was found in four samples (1.57%, 4/255). Among the 187 samples with interpretable Pfkelch13 sequences, four samples presented a mutation (2.1%, 4/187), including one non-synonymous mutation (Y653N) (0.5%, 1/187). This mutation has never been described as associated with artemisinin resistance in Southeast Asia and its in vitro phenotype is unknown. CONCLUSION: This preliminary study indicates the absence of Pfkelch13 mutant associated with artemisinin resistance in Bangui. However, this limited study needs to be extended by collecting samples across the whole country along with the evaluation of in vitro and in vivo phenotype profiles of Pfkelch13 mutant parasites to estimate the risk of artemisinin resistance in the CAR.