Integrated microscopic and molecular diagnosis of imported mixed malaria caused by Plasmodium falciparum and Plasmodium ovalecurtisi: an Italian case report

Abstract Malaria remains a major global health threat, particularly in endemic regions of sub-Saharan Africa. In 2024 alone, an estimated 282 million cases were reported worldwide, with approximately 610,000 associated deaths. Beyond the high disease burden, mixed-species Plasmodium infections represent an additional clinical challenge, as they are frequently underdiagnosed and may significantly complicate both diagnostic accuracy and therapeutic management even in non-endemic countries. We report the case of a 67-year-old man from Guinea who presented to the Emergency Department with high-grade fever and asthenia shortly after returning to Italy; initial microbiological investigations were negative, despite markedly elevated inflammatory markers were diagnosed. The patient reported a previous untreated malaria episode approximately six months earlier, raising suspicion of a novel Plasmodium infection. A comprehensive malaria diagnostic workflow was promptly initiated, integrating rapid immunoenzymatic testing (IE), microscopy, and molecular assays. The IE test suggested Plasmodium falciparum or a mixed infection as result, while loop-mediated isothermal amplification (LAMP-PCR) confirmed the presence of Plasmodium spp DNA. Microscopic examination of stained blood smears revealed typical P. falciparum ring forms and suspected P. ovale trophozoites; so, species-specific real-time PCR confirmed a mixed infection with P. falciparum and P. ovale , supporting prompt antimalarial treatment. Final molecular confirmation was achieved by Sanger sequencing, which identified the Plasmodium ovalecurtisi as co-aetiological agent of disease. The rapid and integrated diagnostic approach enabled timely initiation of appropriate antimalarial therapy with artemether–lumefantrine and primaquine, leading to complete clinical recovery and gene sequencing allowed a correct species confirmation and epidemiological characterization. This case highlights the importance of combining microscopic analysis with advanced molecular diagnosis for an accurate identification of mixed malaria infections even in country and contributes novel epidemiological data through molecular characterization of P. ovalecurtisi in an imported case.