High Prevalence of Plasmodium Infections and Low P. malariae msp-1 Diversity in Beninese Population: Implications for Malaria Surveillance and Vaccine Development

Abstract

Abstract Background Plasmodium malariae, one of the five parasite species causing malaria in humans, though less widespread than P. falciparum , can result in prolonged and persistent infections. This research aims to monitor asymptomatic Plasmodium infections and characterize genetic diversity in the merozoite surface protein (msp-1) of P. malariae in Beninese population. Methods and findings Blood samples collected from 484 asymptomatic participants in Southern Benin underwent analysis for all human Plasmodium species utilizing rapid diagnostic tests (RDT - PfHRP2/LDH), microscopy, nested PCR, and nested real-time qPCR assay. Mono- or mixed infections of P. malariae underwent msp-1 genotyping. The P. malariae msp-1 gene was amplified by a nested PCR, followed by sanger sequencing. Microscopy and RDTs identified infection rates of Plasmodium spp. at 17% and 20%, respectively, whereas nested PCR significantly elevated rates to 38% (P<0.0001). Furthermore, the utilisation of nested qPCR revealed a 12% increase in Plasmodium spp. prevalence among individuals who tested negative using nested PCR. Molecular methods notably enhanced the detection of P. malariae compared to microscopy, with prevalence rates of 7% and 12% using nested PCR and qPCR, respectively (P<0.05). Genetic analysis of msp-1 revealed a high nucleotide sequence similarity (95-100%). The mean pairwise nucleotide divergence (π) of P. malariae was 0.00602. Gender-based analysis indicated a marginal risk of P. malariae infection in females compared to males (RR: 1.3, 95% CI: 0.9 - 8; P = 0.04). Conclusions The present study uncovered a high prevalence of Plasmodium infections, including sub-microscopic P. malariae . Furthermore, our findings demonstrated a low sequence diversity of P. malariae msp-1 compared to other Plasmodium species. This suggests that msp-1 holds promise as a vaccine candidate against P. malariae infection, given its low genetic diversity and highly conserved amino acids. Author Summary Malaria remains a major public health challenge in sub-Saharan Africa, with most research focused on P. falciparum . However, non-falciparum species such as P. malariae are often overlooked despite their ability to cause prolonged and persistent infections. In this study, we investigated the prevalence of Plasmodium infections among 484 asymptomatic individuals in Southern Benin, with a particular focus on P. malariae . Using a combination of rapid diagnostic tests, microscopy, nested PCR, and nested quantitative PCR, we found that molecular methods significantly improved the detection of Plasmodium infections, revealing that P. malariae infections were more common than previously detected by conventional methods. Genetic analysis of the msp-1 gene of P. malariae showed low sequence diversity, indicating highly conserved regions that could serve as potential vaccine targets. Our findings highlight the underestimated prevalence of P. malariae and demonstrate the utility of sensitive molecular tools for detecting sub-microscopic infections. These results provide important insights into the epidemiology and genetic diversity of P. malariae in Benin and support further research into vaccine development and malaria control strategies targeting non-falciparum species