Genetically diverse and multiple Plasmodium falciparum malaria infections occur in asymptomatic individuals within the population (34, 36). The presence of diverse Plasmodium falciparum parasite strains in asymptomatic individuals presents a major challenge to malaria elimination (38). This is because asymptomatic infections serve as reservoirs of malaria parasites, often go undetected by conventional screening programs, and remain untreated, persisting for over a year in an individual (39, 53). Analyzing Plasmodium falciparum genetic profiles provides valuable information on malaria infection outcomes (54), helping elucidate why some patients develop severe disease while others experience a milder form. It also provides essential parasite characteristics required for designing effective intervention strategies to control or prevent the disease (55).
Compared with other microsatellites, microsatellites 313, Poly α, and TA1 were found to be genetically more diverse, supporting the findings of a previous study by Ishengoma et al. (47) in Tanzania. This suggests that in malaria-endemic SSA countries, 313, Poly α, and TA1 may circulate at higher frequencies. The high polymorphism of these microsatellites reflects the high genetic diversity of Plasmodium falciparum parasites within populations (46, 47).
In this study, Plasmodium falciparum genetic diversity was high in both asymptomatic and symptomatic malaria-infected individuals, although it was slightly greater in asymptomatic individuals (He = 0.809, 95% CI: 0.77–0.85; Ne = 7.42, 95% CI: 6.09–8.75) than in symptomatic (uncomplicated and complicated) malaria-infected individuals. This finding suggests that asymptomatic malaria patients present a broader range of parasite genotypes than symptomatic uncomplicated and complicated malaria patients. Similar observations of high Plasmodium falciparum genetic diversity characterized by high allele frequencies have been reported in asymptomatic compared with symptomatic malaria individuals in Cote d'Ivoire (56). Additionally, high Plasmodium falciparum genetic diversity, as indicated by the mean expected heterozygosity (He) of 0.81 (range: 0.57–0.95), has previously been noted among children with asymptomatic malaria infections in Kenya (35). In other studies, symptomatic malaria infections have been indicated to harbor highly diverse Plasmodium falciparum infections (57–59). Conversely, some studies have not reported a difference in Plasmodium falciparum genetic diversity between symptomatic and asymptomatic malaria infections (60, 61). The occurrence of high Plasmodium falciparum genetic diversity in asymptomatic infections may stem from two causes. First, in moderate to high malaria transmission areas, the development of strain-specific immunity may favor asymptomatic carriers as reservoirs for parasite transmission, allowing the continuous introduction of new parasite strains into the host population and thereby promoting genetic diversity and increased parasite fitness (3). Second, asymptomatic infections often persist as low parasite density chronic infections, which can sustain diverse parasite strains within the host (62, 63). In addition to reducing the occurrence of clinical disease, increased genetic diversity within a parasite population increases the risk of antimalarial drug resistance and could lower the efficacy of malaria vaccines (64–66).
The Plasmodium falciparum MOI did not significantly differ between asymptomatic and symptomatic Plasmodium falciparum infections, although it was slightly higher among asymptomatic individuals than among symptomatic malaria individuals. This finding aligns with several previous studies reporting no significant differences in MOI between symptomatic and asymptomatic malaria individuals (37, 56) or between uncomplicated and complicated malaria cases (54). Conversely, other studies have reported higher MOIs in asymptomatic malaria individuals than in symptomatic individuals (67). For example, a recent study by Sarah-Matio, Elangwe M., et al. (34) conducted in a high malaria transmission area in Cameroon revealed a greater MOI in asymptomatic individuals than in symptomatic individuals (MOI = 5 in asymptomatic individuals versus median MOI = 2 in symptomatic individuals, P < 0.001). However, Simpson, S.V. et al. (32) reported a contrasting result, with a higher MOI in symptomatic individuals than in asymptomatic malaria-infected individuals (2.24 versus 1.69; 95% CI 0.01–0.72; p = 0.046). Asymptomatic malaria cases often occur in areas with relatively high transmission intensities (68, 69). In high transmission settings, multigenotype infections result from exposure to multiple parasite strains from different mosquito bites (superinfection) or from a single mosquito carrying multiple strains (co-transmission) (14, 15). Additionally, asymptomatic malaria infection may be associated with partial immunity (53, 70), allowing a wider range of parasite strains to establish infection simultaneously. The presence of multiple parasite strains in asymptomatic malaria infection poses a risk for developing symptomatic malaria (67, 71), in addition to providing a reservoir of genetically diverse parasites (34, 61).
Significant differences were observed in parasite density and hemoglobin (Hb) levels across individuals with asymptomatic and symptomatic malaria (p < 0.001). Symptomatic uncomplicated and complicated malaria patients had higher parasite density and lower Hb levels than asymptomatic malaria patients did. Increased Plasmodium falciparum parasite density has been reported in complicated malaria cases in previous studies (72, 73), whereas low parasitemia has been observed in asymptomatic individuals (74). However, other studies have not reported a significant difference in parasite density between asymptomatic and symptomatic malaria infections (75). Asymptomatic individuals tend to have partial immunity through repeated exposure, allowing them to control parasite levels without experiencing symptoms (19). The reduced parasite density among symptomatic individuals allows them to have high Hb levels compared with symptomatic malaria-infected individuals (76). High parasite density associated with inadequate dosing poses a risk for the development and emergence of antimalarial resistance (77). Additionally, individuals with asymptomatic malaria often do not seek antimalarial treatment, allowing their infections to persist for extended periods of time (78) and serve as a reservoir for malaria transmission (79, 80). Thus, malaria control programs should design strategies to eliminate asymptomatic malaria infections.
The study had several limitations. Genotyping of parasites based on a few selected microsatellites may have resulted in an overestimation of Plasmodium falciparum genetic diversity and MOI, as these markers may not fully represent the true genetic diversity across the entire genome. Therefore, the use of several genetic markers or whole-genome sequencing may provide a more comprehensive assessment of genetic diversity. Additionally, the study included a small number of symptomatic complicated individuals, which might have influenced the accurate evaluation of Plasmodium falciparum genetic diversity and the MOI in that group.