Malaria transmission and management strategies are affected by the genetic diversity of P. falciparum parasites (Chen et al., 2018). Understanding P. falciparum genetic diversity in different geographical settings is critical for determining the parasite's population structure and designing effective control strategies (Mohd Abd Razak et al., 2016). Although significant efforts have been made to control and eventually eliminate malaria in Ethiopia, there are limited molecular data on P. falciparum genetic polymorphisms, which are the most prevalent and virulent malaria parasites in the region. This study assessed polymorphisms of the merozoite surface protein-2 gene in samples from different study sites in Northwest Ethiopia. The population of P. falciparum isolates from the study area had significant genetic diversity.
The isolates with MSP 2 diversity had a high prevalence of IC/3D7 allelic families, according to this investigation. This suggests that these allelic families are better adapted to their host in the study setting than FC27. The prevalence of dominant alleles is caused by genetic recombination, which results in parasites with high frequencies of certain alleles and, consequently, a high level of biological fitness (Miotto et al., 2013). Furthermore, the predominance of the IC/3D7 allelic family could be attributed to balancing selection acting on these allelic variants (Metoh et al., 2020). This finding is also consistent with reports from South Benin (Ogouyèmi-Hounto et al., 2013), Bobo-Dioulasso, Burkina Faso (Somé et al., 2018), Pahang, Malaysia (Atroosh et al., 2011), and Adama, Ethiopia (File et al., 2022) but differs from genotyping in the Chewaka district, Ethiopia (Abamecha et al., 2020), Kosti, central Sudan (Hamid et al., 2013) and Khyber Pakhtunkhwa, Pakistan (Ullah et al., 2022). This implies that the allelic family of P. falciparum could vary based on geographical location, transmission intensity, and the size of the study population (Mohammed et al., 2017).
A total of 38 different alleles for MSP 2 were obtained from the parasite isolates in this study area. The presence of multiple parasite populations results in the mixing of genotypes, leading to increased genetic diversity (Mohammed et al., 2019). This genetic diversity was greater than the diversity found in Humera, North West Ethiopia (MSP2:12) in 2018 (Mohammed et al., 2018), Melka-Werer, Northeast Ethiopia (MSP2:14) in 2021 (Mohammed et al., 2021), Kosti, Central Sudan (MSP2:16) in 2013 (Hamid et al., 2013), but lower than that in China (MSP2:52) in 2019 (Zhang et al., 2019) and North Central Nigeria (MSP2:54) in 2021 (Usman-Yamman et al., 2021). This implies that P. falciparum infection diversity could vary depending on geographical location, transmission intensity, malaria treatment policies, and sample population (Khatoon et al., 2010, Mohammed et al., 2017). The high level of genetic diversity observed in parasite populations in high transmission areas might be due to genetic recombination (Duah et al., 2016). During genetic recombination, novel combinations of alleles with beneficial features for the parasite can be generated through positive selection, which enables alleles to spread through the population (Metoh et al., 2020). Similarly, more malaria-endemic sites had greater genetic diversity than lower malaria-endemic sites (Kateera et al., 2016). This shows that the level of endemicity determines P. falciparum genetic diversity. Similarly, treatment policies that focus on the use of highly potent drugs that target both asexual blood-stage parasites and gametocytes are more effective than drugs that act only on blood-stage parasites. Highly potent drugs effectively decrease parasite transmission and clonal diversity (Greenwood et al., 2008, Targett et al., 2001). The reduction in parasite transmission due to effective drugs may have a significant impact on the parasite's genetic variability, which may affect its adaptive ability and opportunities for sexual recombination (Laufer and Plowe, 2004).
The heterozygosity in the present study was high (He = 0.948), which indicates a wide range of allele diversity within the msp2 genes, which is greater than that previously reported in Ethiopia (Mohammed et al., 2017, Mohammed et al., 2021, Mohammed et al., 2018). This indicates a high transmission pattern of distinct P. falciparum clones in the study area. The presence of various P. falciparum clones is indicated by the circulation of various fragment sizes of MSP 2 alleles (Oboh et al., 2021). Genetically different clones may adapt better to existing interventions and increase the likelihood of developing antimalarial resistance (Amoah et al., 2021).
This study revealed a significant level of multiclonal infection in MSP 2 genotypes. This can be the result of multiple infectious mosquito bites or the transmission of genetically diverse sporozoite inoculum from a single mosquito bite (Metoh et al., 2020). The results of this research demonstrated that 96.7% of the isolates contained multiple genotypes. This shows that the P. falciparum parasite population in this area is very diverse. The high rate of mixed infections in the studied area implies a high degree of transmission. This finding is similar to reports from South Benin (Ogouyèmi-Hounto et al., 2013) and Pawe District, Northwest Ethiopia (Mohammed et al., 2017), but higher than those of a previous study in the Kolla-Shele region of Arbaminch Zuria District in Ethiopia (Mohammed et al., 2015) and Humera, Northwest Ethiopia (Mohammed et al., 2018).
The overall multiplicity of infection (MOI) was 3.46. This was higher than that reported in studies conducted in China (Zhang et al., 2019), Bobo-Dioulasso, Burkina Faso (Somé et al., 2018), Northeast India (Sathishkumar et al., 2022), North Central Nigeria (Usman-Yamman et al., 2021), Adama, Ethiopia (File et al., 2022), and Humera, Northwest Ethiopia (Mohammed et al., 2018), but lower than that reported in West Benegal, India (Saha et al., 2016) and South Benin (Ogouyèmi-Hounto et al., 2013). The variation in the multiplicity of infection could be due to the level of malaria endemicity, malaria transmission pattern, and other factors, such as the age of the study population, parasite density in the study population, use of different antimalarial drugs, and immune status (Chen et al., 2018, Ullah et al., 2022, Mayengue et al., 2011). The multiplicity of infection (MOI) is generally assumed to be greater in high malaria transmission settings, but in low malaria transmission settings, most individuals can carry single-clone infections (Lopez and Koepfli, 2021). The greater the level of malaria transmission is, the greater the chance of having a higher MOI and mean number of alleles per locus. Similarly, more effective antimalarial drug therapy is expected to reduce the number of parasite strains surviving treatment, reducing the strain multiplicity in the parasite population (Orwa et al., 2019). However, high levels of infection complexity were detected in this study, implying that any possible contribution of drugs to reducing the observed diversity may have been insignificant (Khatoon et al., 2010). In the same vein, the immune status of the patient influences the parasite multiplicity of infection. The high levels of multiple clones in the samples reflect a low level of acquired immunity and a consequently poor capacity to control the infection (Ogouyèmi-Hounto et al., 2013).
The findings of this study show that age has no association with multiple infections. The results of this study are supported by previous findings from Pakistan (Khan et al., 2021), the Kolla-Shele region of the Arbaminch Zuria District in Ethiopia (Mohammed et al., 2015), Brazzaville, the Republic of the Congo (Mayengue et al., 2011), and North Central Nigeria (Usman-Yamman et al., 2021) but contrast with reports from Bioko Island, Equatorial Guinea (Chen et al., 2018). This shows that the MOI is not directly related to the duration of immune acquisition in asymptomatic patients but reflects the exposure of subjects to malaria in the endemic area (Ullah et al., 2022).
The findings of this study also suggest that younger age groups had relatively high parasite densities and multiple parasite populations. This may be due to being less likely to resist parasitic infection due to low immunity (Rono et al., 2013).
The multiplicity of infection in this study was significantly influenced by parasite density. There are many reports demonstrating that high parasite densities increase the probability of detecting concurrent clones in an individual (Salem et al., 2014, Kateera et al., 2016). These findings agreed with those of Khyber Pakhtunkhwa, Pakistan (Ullah et al., 2022), Bioko Island, Equatorial Guinea (Chen et al., 2018), Brazzaville, Republic of Congo (Mayengue et al., 2011), Kosti, Central Sudan (Hamid et al., 2013), Adama, and Ethiopia (File et al., 2022) and were negatively correlated with those of Melka-Werer, Northeast Ethiopia (Mohammed et al., 2021). This finding shows that P. falciparum genetic diversity could be correlated with a high parasite density in the study area. The limitations of this study include the use of a single blood sample, which may not be enough to show the whole diversity of parasites carried by an individual since genotypes can appear and disappear in a very short time. Additionally, the genotyping of P. falciparum isolates using a single antigenic marker may underestimate the diversity of infections. This study was unable to explore the relationships between malaria severity or clinical symptoms and genetic diversity. Furthermore, the coinfection statuses of other diseases were not screened, and the influence of the patient's immune status on the genetic diversity and multiplicity of infection of the parasite was not addressed. The study could also not exclude individuals receiving antimalarial therapy, which can reduce the multiplicity of infections.