Molecular typing reveals sub-microscopic P. ovale and P. malariae infections associated with anemia among asymptomatic populations in the Sudano-Sahelian region of Chad

Unravelling malaria parasite species composition is critical for malaria elimination, especially in the Sudano-Sahelian seasonal transmission geography. Here, we employed a suite of RDT, microscopy, and PCR on 601 blood samples collected between November 2024 and January 2025 in a community-based cross-sectional study to examine the local Plasmodium species and assess the associated clinical impact on hemoglobin level in a setting characterized by intensive deployment of malaria control interventions. The findings reveal an RDT, microscopy and PCR-positive malaria prevalence of 23.6% (142/601), 28.1% (169/601), and 57.6% (346/601), respectively. Among PCR-detected Plasmodium infections, P. falciparum mono-infection was most common [58.1% (201/346)]. Sub-patent non-falciparum mono-infections included P. malariae [2.6% (9/346)] and P. ovale species [2.0% (7/346)]. Multi-species infections occurred at varying frequency, including P. falciparum/P. malariae [11.3% (39/346)], P. falciparum/P. ovale [17.6% (61/346)], P. malariae/P. ovale [3.2% (11/346)], and P. falciparum / P. malariae / P. ovale [5.2% (18/346)]. Co-infections were predominant among the 6–15 years and > 40 years age groups [OR = 7.3 (95% CI 4.1–11.5), p = 0.004]. Hemoglobin concentration was significantly lower (Welch's independent-samples t-test: mean difference = − 2.29 g/dL; 95% CI − 2.51 to − 2.08; p < 0.001) among Plasmodium -infected individuals [n = 346; mean Hb = 9.58 ± 1.40 g/dL] compared with non-infected individuals [n = 255; mean Hb = 12.6 ± 1.29 g/dL]. Non-falciparum infections (comprising P. malariae and P. ovale mono-infections and P. malariae + P. ovale co-infections) were associated with moderate anemia [Hb level = 9.3 g/dL; OR = 3.4, (95% CI 1.6–7.2), p = 0.03]. Further molecular typing revealed the circulation of P. ovale curtisi (36/70; 51.4%) and P. ovale wallikeri (19/70; 27.1%) sibling subspecies, and sequence alignment of the merozoite surface protein-1 gene of nine local Chadian P. malariae isolates indicates probable low genetic diversity (π = 0.002, Hd = 0.72) and phylogenetic kinship with the Cameroon historical reference strain, suggesting geographical relatedness with potential implications on cross-border malaria control efforts. Cumulatively, our study underscores the silent circulation of non-falciparum species and provides evidence to the Chad National Malaria Control Programme for policy reforms on a comprehensive public health strategy that extends beyond P. falciparum to include all relevant malaria parasite species towards the elimination goal.