The hidden malaria: misidentification of Plasmodium malariae and Plasmodium ovale in low-transmission setting targeted for elimination in Ethiopia

Abstract Background Quartan malaria caused by Plasmodium malariae (Pm) and tertian Plasmodium ovale (Po) malaria has been largely overlooked in Ethiopia due to their milder clinical symptoms, lower prevalence, and frequently misdiagnosis during routine microscopic. This study estimated the prevalence of Pm and Po infections misdiagnosed as P. vivax (Pv) in selected health facilities in southwestern Ethiopia. Methods A cross-sectional study was conducted among study participants who were positive for Pv by microscopy or rapid diagnostic test (RDT) and who were attending health facilities within Arjo-Didessa sugarcane plantations and surrounding areas in Oromia, southwestern Ethiopia, between September 2019 and July 2022. Of the 3590 febrile patients attending health facilities, 323 were Pv positive by microscopy or RDT. Among these, 245 Pv-positive samples were re-evaluated using quantitative polymerase chain reaction (qPCR). Species-specific primers targeting Pv, P. falciparum (Pf), Pm, and Po were used for species confirmation. Results From 245 vivax malaria cases, RDT were performed to 162 sub-samples, among them 8.6% (14/162) were negative by RDT. None of the samples tested by RDT and microscopy detected Pm or Po. Of these 245 tested and reported as Pv positive by microscopy, qPCR confirmed that 215 (87.7%, 95% CI 82.98–91.58) were positive for Pv, 9 (3.7%, 95% CI 1.69–6.86) for Pf, 8 (3.3%, 95% CI 1.42–6.33) for Pm, 6 (2.4%, 95% CI 0.90–5.25) were positive for Po, and 7 (2.8%, 95% CI 1.16–5.80) had mixed infections (Pf and Pv). Conclusions The findings of this study highlights the need for a strategic shift to integrate Pm and Po into Ethiopia's malaria control and elimination program. Moreover, strengthening the diagnostic capacity should include training of microscopists for accurate Plasmodium species identification and adopting highly sensitive diagnostic tool for all four Plasmodium species in resource-limited settings. This is essential for enhancing malaria control and accelerating progress toward elimination.