Implementation and validation of a new qPCR assay to detect imported human <i>Plasmodium</i> species

Diagnosis of imported malaria is based on microscopic examination of blood smears (BS), detection of circulating plasmodial antigen by immunochromatography (ICT), or detection of <i>Plasmodium</i> spp. DNA by loop mediated isothermal amplification. We have developed duplex (<i>Plasmodium</i> spp. and <i>Plasmodium falciparum</i>) real-time PCR (qPCR) and species-specific qPCR assays for the identification and quantification of human <i>Plasmodium</i> species. Whole nucleic acids from 523 samples prospectively collected from 410 patients suspected of malaria between June 2016 and March 2021 were tested by qPCRs and compared to standard diagnostic procedures. All qPCRs were designed on 18S ribosomal ribonucleic acid. The limit of detection of duplex qPCR was 8 copies/reaction, and analytical specificity of species-specific qPCRs was 100%. Seventy-nine patients diagnosed for single species malaria involving <i>P. falciparum</i>, <i>P. vivax</i>, <i>P. ovale,</i> and <i>P. malariae</i> were positive with duplex and species-specific qPCRs. <i>P. knowlesi</i> qPCR detected DNA from a <i>P. knowlesi</i> culture. Of eight cases of mixed <i>Plasmodium</i> species infection, five were identified with our qPCR assays with better sensitivity as compared to BS and ICT. Eight out of 323 patients with negative BS were hospitalized for symptoms suggestive malaria after malaria-endemic area travel or known with history of malaria had a low positive duplex qPCR. Between day of diagnosis and post-treatment follow-up at D2-D4 of malaria, a 3.1-log <i>P. falciparum</i> load decrease was observed by qPCR. These new <i>Plasmodium</i> qPCRs allowing detection of human plasmodial species have excellent species specificity and allow rapid detection of <i>P. falciparum</i> cases, malaria with low parasitaemia, and mixed <i>Plasmodium</i> species infection.IMPORTANCEMalaria is a disease transmitted by a <i>Plasmodium</i> parasite genus. Most cases are caused by <i>Plasmodium falciparum</i>. Despite a significant drop of incidence and mortality since 2000, 249million cases and 608,000 deaths have been estimated in 2022, mainly in Africa. Due to the increasing number of travels to endemic areas, incidence of imported malaria is rising in Europe. Various techniques are used in European laboratories, such as microscopic examination of thin and thick smears and rapid diagnostic tests. However, these techniques require skilled operators to differentiate plasmodial species and have limited sensitivity. Actually, molecular diagnosis is carried out using point-of-care test for rapid results with excellent sensitivity but is unabled to determine involved species and assess parasitaemia. In this study, we developed a combined molecular tool based on both detection of all human plasmodial species (<i>Plasmodium</i> spp.) and <i>P. falciparum</i>. We have also developed specific qPCRs for each human plasmodial species.