Underappreciated Transmission of Plasmodium malariae and Plasmodium ovale: A Potential Barrier to Malaria Elimination in Bangladesh

Plasmodium malariae ( P. malariae ) and Plasmodium ovale ( P. ovale ) spp. are underappreciated contributors to residual malaria transmission, especially in near-elimination settings. They remain absent from routine surveillance in Bangladesh, where field diagnosis relies on Plasmodium falciparum ( P. falciparum )/ Plasmodium vivax ( P. vivax )-specific rapid diagnostic tests (RDTs) and microscopy. Cases escaping detection by ongoing malaria surveillance in Bandarban, Bangladesh, were investigated in the present study. Blood samples collected in 2025 from febrile patients that tested negative for P. falciparum/P. vivax via RDT underwent microscopic examination and molecular testing. Genomic DNA was screened using a SYBR-Green-based real-time quantitative polymerase chain reaction (qPCR) test targeting the 18S ribosomal RNA (rRNA) gene. Plasmodium ovale- positive cases were confirmed via a probe-based qPCR assay, with P. ovale curtisi and P. ovale wallikeri identified using a validated assay targeting small subunit rRNA and Po-ldh genes. Archived laboratory records (2014–2024) were reviewed to identify previously detected P. malariae infections. In 2025, five P. ovale spp. (four P. ovale curtisi and one P. ovale wallikeri) and three P. malariae cases were detected in Alikadam Subdistrict, Bandarban. All cases were RDT-negative despite microscopically detectable parasitemia (mean 4,536 parasites/ µ L for P. ovale and 6,053 parasites/ µ L for P. malariae ). Field microscopy was used to correctly diagnose only one P. ovale case, with the remaining four being misclassified as P. vivax or mixed P. malariae / P. vivax infections. Analysis of archived data revealed 25 additional qPCR-confirmed P. malariae infections. Cases were detected across multiple ethnic communities, predominantly among adult males in forest-adjacent settlements. Integrating pan-species molecular diagnostics into routine surveillance, along with species-specific case reporting, is essential to effectively detect, monitor, and eliminate these underappreciated malaria species.