Uncovering silent malaria reservoirs in Southern Ethiopia: community-based molecular detection of asymptomatic and sub-microscopic infections and their implications for diagnosis and control

Abstract

BACKGROUND: Prompt and accurate detection of asymptomatic and sub-microscopic malaria infections remains a critical challenge for routine malaria diagnosis and control. Asymptomatic individuals rarely seek medical care and are often missed by standard diagnostic methods. When combined with low-density parasitemia, these undetected infections persist, sustaining and fueling ongoing residual malaria transmission. This study aimed to investigate the presence of asymptomatic and sub-microscopic Plasmodium infections. METHODS: A cross-sectional community-based study was conducted from September to December, 2024, enrolling 525 participants across ten representative kebeles from five woredas by using multi-stage sampling in the Gedeo Zone. Peripheral blood samples and dried blood spots were collected and screened for malaria parasites using microscopy, rapid diagnostic tests, and quantitative polymerase chain reaction. Data were coded, entered into Epidata version 4.6, cleaned up, and analyzed using SPSS version 25 to estimate the prevalence of asymptomatic and submicroscopic infections and their associated demographic factors. RESULTS: qPCR detected a substantially higher prevalence of asymptomatic 164/525 (31.2%) and sub-microscopic infection 72/433 (16.6%) compared to microscopy asymptomatic infection 92/525 (17.5%) and RDT asymptomatic infection 69/525 (13.1%) prevalence. Of these sub-microscopic infections, 34/433 (7.9%) were P. vivax, 31/433 (7.2%) were P. falciparum, and 7/433 (1.6%) were mixed infections. These sub-microscopic infections were more common among participants aged 15-24 years and among female study participants. CONCLUSION: Asymptomatic and submicroscopic Plasmodium infections, prevalent yet undetected by microscopy and RDTs, were reliably identified by qPCR, revealing substantial underestimation of true prevalence and a persistent transmission reservoir that challenges malaria control. These results underscore the limitations of symptom-based strategies and the need for sensitive diagnostics to enhance surveillance of low-density infections.