Persistence of Plasmodium falciparum DNA detection after antimalarial treatment and the implication of gametocytes: a prospective cohort study in a non-endemic setting

Vollständiger Abstract

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ABSTRACT Highly sensitive molecular assays are increasingly used for malaria diagnosis but are not recommended for follow-up, as Plasmodium falciparum DNA detection may persist after effective antimalarial treatment, complicating these tests’ clinical interpretation. The biological basis of this persistence remains poorly defined. We investigated the potential role of gametocytes in the context of treated infections. In a prospective cohort of 100 successfully treated patients, we analyzed 379 blood samples collected up to day 28 (D28) post-treatment. We used quantitative PCR (qPCR) to detect parasite DNA targeting PfvarATS , a mainly asexual-stage marker, and 18S ribosomal RNA ( 18S rRNA ), a pan-stage marker. To differentiate residual nucleic acid detection from ongoing parasite transcriptional activity among patients’ samples that remained DNA-positive at D28, we used RT-qPCR targeting PfvarATS , 18S rRNA , and gametocyte-specific transcripts ( PfAP2-G , Pfs16 , PfMGET , and PfCCP4 ). After anti-malarial treatment, 61% of samples remained qPCR positive at D28. PfvarATS transcripts declined sharply (12% positivity rate at D28), while 18S rRNA transcripts persisted longer (60% at D28). Gametocyte-specific markers were detected in a subset: Pfs16 (immature stages) was frequent early (100% at D0 and 94% at D3), while mature gametocyte markers ( PfCCP4/PfMGET ) peaked at D7 (33%) and were still detectable in 12% of samples at D28. Therefore, most D28 PCR positivity (88%) occurred without detectable mature gametocyte transcripts. Persistent P. falciparum DNA detection post-treatment in a non-endemic setting predominantly reflects non-viable parasites or residual genetic material and a minority of circulating mature gametocytes. This highlights a key diagnostic pitfall: post-treatment PCR positivity does not equate to treatment failure. Results must be interpreted with clinical context, timing, and assay targets. IMPORTANCE This study confirmed that persistent P. falciparum DNA detection after effective antimalarial treatment mainly reflects residual parasite material, challenging the use of PCR in treatment efficacy follow-up. By excluding mature gametocytes as the primary source of persistent DNA detection, this study advances our understanding of post-treatment parasite nucleic acid kinetics in the organism and supports the importance of investigating alternative biological mechanisms.