The genetic diversity of P. falciparum parasites impacts malaria transmission and malaria control strategies [53]. Genetic structures and population genetics studies of P. falciparum may hold the key for effective disease surveillance and control programmes, especially in Southwest Ethiopia as so far there is very limited information available on the genetic structures of P. falciparum. As the country moves towards malaria elimination, understanding the genetic diversity and population structure of the malaria parasite populations in hotspots is crucial to guide monitoring and evaluation of malaria control strategies and anti-malarial interventions. The present study provides a detailed assessment of genetic diversity and multiplicity of infection of P. falciparum parasites from Chewaka district, Southwest Ethiopia.
In this study, allele-specific PCR typing of the msp-1 and msp-2 loci showed considerably diverse and extensive allelic polymorphisms in P. falciparum populations in the analysed samples. However, the number of alleles may have been underestimated due to the limitations of the technique used. Indeed, the numbers of alleles (bands) detected may be underestimated due to sensitivity of the PCR technique used as minor fragments (<50 bp) cannot be detected on the agarose gel and also similar sized fragments may be classified as identical leading to a false impression of similarity. Within allele families, alleles of the same size may have different amino acids motifs [51, 52], which emphasizes the importance of sequencing in future studies to confirm diversity and extensive allelic polymorphisms in the P. falciparum. A total of 10 and 15 different alleles for msp-1 and msp-2, respectively, were obtained from the parasite isolates in Chewaka district, Ethiopia. This genetic diversity was consistent with the diversity found in Kolla-Shele area, Southwest Ethiopia (msp-1: 11; msp-2: 12) in 2015 [42], in Northwest Ethiopia (msp-1: 12; msp-2: 22) in 2018 [43], and Brazzaville in the Republic of Congo (msp-1: 15; msp-2: 20) in 2018 [54]. In contrast, a higher diversity (msp-1: 26; msp-2: 25) was found in Bioko Island, Equatorial Guinea in 2018, even though this area has comparable malaria endemicity patterns [53]. K1 was the predominant allelic family for msp-1 as also demonstrated in previous studies in Africa, including Southwest Ethiopia [42], Brazzaville, Republic of Congo [16] and Gabon [55]. However, in studies conducted in Northern Ethiopia [44], Central Sudan [56] and Bioko Island, Equatorial Guinea [53] the MAD-20 allele was found to be predominant.
In this study, the RO33 family showed no polymorphism with only a single allele (160bp). This is similar to findings in Congo [16]. Allele typing of msp-2 showed that FC27 was the predominant allelic family as also demonstrated in previous reports from Benin [57] and Central Sudan [56], but in contrast with previous studies in Ethiopia [42] and Brazzaville, Republic of Congo [16]. A variation in the prevalence of alleles between different studies likely reflects the differences in sample population. Thus, it is important to conduct studies that include adequate sample size as well as sampling at different time point within the same region to assess and compare the genetic profile of parasites circulating in endemic areas in an attempt to avoid intra and inter individual variation in the number of parasite genotypes detected in the different episodes of malaria. Besides, methodological differences may also affect the comparability of results. Hence, further investigations with more powerful techniques such as capillary electrophoresis and DNA sequencing are needed to better characterize the malaria parasites in the country.
Multiplicity of infection (MOI), i.e. the number of different P. falciparum strains co-infecting a single host, has been shown to be a common feature in most malaria-endemic areas and was reported to vary with age, parasite density, immune status, epidemiological settings and transmission intensity [58, 59, 60, 61]. In this study, 80% of the isolates harboured more than one parasite genotype identified by the presence of two or more alleles of one or both genes with the overall mean MOI being 3.2 (95% CI: 2.87- 3.46). The overall MOI value reported in this study was higher than previously reported studies, including Ethiopia (MOI: 1.8 - 2.6) between 2015-2018 [42, 43, 44], Brazzaville, Republic of Congo (MOI: 2.2) [16] in 2011 and Bobo-Dioulasso, Burkina Faso (MOI: 1.95) [62]. In contrast to study reported in Bioko Island, Equatorial Guinea (MOI: 5.51) [53] in 2018 and Gabon (MOI: 4.0) [63] in 2018. The difference in MOI can be explained by the differences in intensity of malaria transmission seasons. In this study, samples were collected during the major malaria transmission season of September to December, when malaria transmission is very intense. All year round (seasonal) studies covering major and minor transmission seasons are needed to better understand genetic profiles in this area including a sense on seasonal variations.
The results of this study show that age has no association on multiplicity of infection similar to other studies [42, 44, 50], but in contrast with reports from Brazzaville, Republic of Congo [53] and Central Sudan [56]. Previous studies regarding the variation of MOI over age have suggested that the influence of age on the multiplicity of infection is highly affected by endemicity of malaria [57, 58, 59, 60]. This is probably a reflection of the development of anti-parasite specific immunity [31]. Thus, in holo- or hyperendemic areas, immunity develops faster and at younger age than in areas with less intense transmission [64]. Studies have shown an age-dependent MOI in a village with intense perennial malaria transmission but not in areas where malaria is mesoendemic [50, 59]. Similarly, in this study reported that no significant relation between MOI and the parasite count, similar to reports from previous studies in Ethiopia [42, 44], but in contrast with reports from Bioko Island, Equatorial Guinea [57]. This may have been due to the small number of isolates analysed.
High transmission regions like those in many African countries are commonly characterized by P. falciparum populations that are genetically diverse. Antigenic marker genotyping carried out in African regions like Burkina Faso, Sao Tome, Malawi, Uganda and Tanzania have identified P. falciparum populations with alleles occurring at a frequency below 10 percent with a very high He level of 0.78 to 0.99 [17]. This study indicate that the genetic diversity values were higher based on heterozygosity index for msp-2 (He=0.85), than for msp-1 (He=0.43), suggesting a large genotype diversity within the msp-2 locus, which was higher than previously reported from Northwest Ethiopia (msp-2: He 0.62) in 2018 [44]. Djibouti, a neighbouring country to Ethiopia, an initially moderate level of genetic diversity declined over an 11-year period to the point that the expected heterozygosity reached zero in 2009 consistent with very low diversity [30].
Despite the lack of entomological data from Chewaka district, the number of clones co-infecting a single host can be used as an indicator of the level of malaria transmission or the level of host acquired immunity [58, 65, 66]. Besides, transmission intensity can also be affected by other factors, such as vector biting behaviour and endemicity [65]. Inferring high transmission intensity from the presence of multi-clonal infections alone has additional limitations including estimates of MOI varying by genotyping method, potential impact from sampling frequency and a non-linear relationship between MOI and transmission intensity [65]. Despite these limitations, infections with multiple clones observed in this study, combined with evidence of high genetic diversity may indicate high transmission intensity in the study area.
Limitations of the study
The limitation of the present study is the small number of isolates analysed, which were collected during a therapeutic efficacy study of artemether-lumefantrine (Coartem®) in the region. In our study, we did not examine the association between the dominant allelic families and the manifestation of the disease because all samples were collected from uncomplicated malaria patients. Thus, the relationship between malaria severity or clinical symptom and genetic diversity could not be addressed in the present study. The collection of samples throughout the year (not just in high transmission season) would potentially give a better understanding of the true diversity in the region. Despite these limitations, the data from the present study has confirmed the high genetic diversity profile and mixed-strain infections of P. falciparum populations in Chewaka district, Ethiopia, potentially reflecting both the endemicity level as well as the fact that malaria transmission remains high in Southwest, Ethiopia.