Malaria is the most significant parasitic infection in humans worldwide, particularly among young children in endemic regions, affecting an estimated 263 million people in 2023, including 9.56 million in Ethiopia [1]. Mixed malaria infections, also termed malaria co-infections, occur when patients are simultaneously infected with multiple Plasmodium species and are more common in regions endemic to two or more species [2,3]. The incidence of mixed malaria ranges from <1 % to 63 % depending on the region [[4], [5], [6], [7]]. In Ethiopia, mixed infections are observed in up to 5 % of cases [[8], [9], [10], [11], [12]]. Mixed infections underscore the need for accurate diagnostic testing, as treatment guidelines vary according to the infecting Plasmodium species [13]. Uncomplicated P. falciparum malaria is treated with artemisinin-based combination therapies, such as artemether-lumefantrine, whereas Plasmodium vivax and Plasmodium ovale infections require anti-relapse treatment with primaquine or tafenoquine, although primaquine failure has been reported [[13], [14], [15], [16], [17]].

Quick, low-cost, and user-friendly rapid diagnostic tests (RDT) are common in low-resource settings but are prone to false-negative results in patients with low parasitemia (<1000 parasites/μL) [[18], [19], [20]]. Microscopy-based blood smears remain the gold standard diagnostic test, although mixed malaria infections are detected by microscopy in only 2 %–17 % of cases [[21], [22], [23]]. Polymerase chain reaction (PCR) offers high sensitivity and specificity in confirming the causative Plasmodium species and is less dependent on technician expertise; however, PCR may yield false positives because it cannot distinguish active parasitemia from recently treated infections [[24], [25], [26]].

Here, we present two cases of malaria in pediatric siblings who recently traveled to Ethiopia, in which PCR failed to detect P. vivax at hospital admission. One sibling also experienced therapeutic failure of primaquine, presumably due to recrudescence without re-exposure. This case demonstrates limitations of both traditional and newer malaria diagnostics and underscores the need to maintain a high index of suspicion for co-infections with strains that may relapse (P. vivax, P. ovale).