Background
Despite an enormous effort to control and eventually eliminate malaria, studies reveal that it is still a major public health problem, especially in sub- Saharan Africa (SSA) where more than 90% of the disease burden prevails [
1,
2]. About 68% of Ethiopian population inhabits in 75% of the countries land mass that is malarious, where
Plasmodium falciparum and
Plasmodium vivax accounts for 70% and 30%, respectively [
3]. Studies revealed that, multiple factors greatly affected malaria control and elimination efforts. From which the frequent emergence and spread of genetic diversity of
P. falciparum is prominent. High genetic diversity is not only an indicator of its evolutionary success [
4] but also, the intensity of transmission [
5] that pose potential challenges in malaria control programmes [
6].
Molecular characterization of
P. falciparum enables us to investigate the genetic diversity of infection with consideration of various factors, such as disease phenotype, age and host immunity [
7]. Genetic diversity of
P. falciparum is usually determined through genotyping of the polymorphic regions block 2 of
msp-1 [
6,
8,
9]. MSP1 is involved in erythrocyte invasion and is one of the major
P. falciparum blood-stage malaria vaccine targets [
10‐
12]. MSP1 is a 190 KDa surface protein encoded by the
msp1 gene located on chromosome 9 and contains 17 blocks of sequences flanked by conserved regions [
9,
13,
14]. The precise functional role of
msp1 during invasion has not been fully evaluated, and its macromolecular characterization is incomplete [
15].
msp-1 markers are useful to investigate genetic diversity, multiplicity of infection (MOI) and parasite carriage. Polymorphism in msp1 and msp2 have been frequently reported from different parts of the world. Of the 17 blocks of msp1, block 2 is the most polymorphic region characterized into three allelic families (K1, MDA20 and R033). Based on the variation in length and sequence diversity, this region is a commonly targeted part in determining genetic diversity and MOI in clinical isolates of P. falciparum.
Even though genetic diversity of P. falciparum has been extensively studied in different parts of the world, limited data are available from Ethiopia. The aim of this study was, to assess genetic diversity of block 2 region of msp-1 gene of P. falciparum clinical isolates from three districts in central Ethiopia.
Discussion
In Ethiopia, even though considerable efforts have been made at national and local levels to control and eventually eliminate malaria, limited molecular data exists on genetic polymorphism of P. falciparum, the most predominant and virulent malaria parasite in the region. The present study aimed to assess the genetic polymorphism of P. falciparum clinical isolates based on block 2 region msp-1 genotypes and multiplicity of infection. This is the first study that widely investigated the status of P. falciparum genetic diversity from three districts of the study areas in central Ethiopia. Moreover, the study examined the spatial and seasonality of such polymorphism in relation to parasite density and other patient characteristics.
The study revealed that; geometric mean of parasite density was disproportionately high in school age children (SAC) and relatively stable afterwards (Fig.
2). In addition, there was no statistically significant correlation existed between parasite density and age of the patients (Pearson’s correlation = 0.12, P = 0.6). Even though a number of factors may contribute to the fluctuation of parasitaemia level overtime in symptomatic patients, the geometric mean of microscopically detectable parasitaemia levels could be used to explain the finding of this study [
24]. The major factor that mainly contributed for higher parasitaemia level in SAC is delayed acquisition of protective immunity during this immunological transition age making this age group more vulnerable to malaria infection than adults [
25].
In the present study, multiple infections slightly increased with age group (Fig.
2B), although the variation was not statistically significant (X
2 = 0.5). This finding is in congruent with the report from Burkina Faso [
26] and Tanzania [
27], where they explained that episodes of infection in children is commonly for very short duration and the duration of episodes of infection increases with age contributing to the multiple infections. Other reports suggested that multiple infections vary with parasite density, immunity status, the overall prevalence of infection in the population and transmission intensity as reviewed by [
28‐
30]. Other studies have shown an inverse association. Therefore, the relationship between malaria patient age, level of parasitaemia, number of clones of infection, transmission intensity and status of immunity to malaria parasite needs further investigation.
In the present study, there was no significant correlation existed between multiple clone infections of
P. falciparum with seasonal variation of malaria incidence and travel history of patients (Table
3). In favour of this finding, report from southwestern Ethiopia [
31], has shown the absence of correlation or negative correlation between the proportion of multi-clonal infections and parasite prevalence. On the other hand, reports from Indonesia [
32], and Papua New Guinea [
33], show the presence of positive correlation between the rate of polyclonal infections and annual parasite incidence. The predominance of polyclonarity (92%) in those patients having no travel history depicts real features of malaria epidemiology with respect to the genetic marker of
msp-1 gene in the study area.
In this study, 26% of the isolates having multiple genotype infections. The overall MOI of 1.3 and the expected heterozygosity of 0.39 (Table
2). This finding differs from north western Ethiopia and southwestern Ethiopia reported by Mohammed et al
. [
23] and Abamecha et al
. [
34] with 75% and 80% frequency of multi-clonal infections, and 1.8 MOI with
He (0.79), 2.0 MOI and
He (0.43), respectively. This shows that malaria transmission in the study under report exhibits slightly low genetic diversity, compared with northwestern and southwestern Ethiopia. This could be due the locational advantage of central Ethiopia to better health services, differences in local epidemiology, demographic and environmental conditions that might have resulted in observed reduced genetic diversity pattern in Adama and its surroundings. In the present study, from 139 samples 19 different length polymorphism of
msp-1 allelic variant was revealed; 8 MAD20 (160–330 bp), 6 K-1 (100–270) bp, and 5 RO33 (100–220 bp). This shows the level of size polymorphism of
msp-1 alleles in the study area. However, the number of alleles identified may have been under estimated due to a number of limitations like sensitivity of PCR technique used, inability to differentiate minor fragments, the possible existence of similar size fragments and the same size fragment having different amino acid motifs [
34,
35].
Size polymorphism of
msp-1 allelic variant identified in the present study is slightly higher than the report from Chewaka district of southwestern Ethiopia [
34] and Humera of north-western Ethiopia [
6]. This was less diverse than Kolla Shele district of south western part of Ethiopia [
23], but more or less similar to reports from Equatorial Guinea [
22], and Bobo-Dioulasso in Burkina Faso [
36]. The major factor that may account for such variation could be the scope of study sites covered and local malaria transmission patterns might have contributed. Gel- analysis of the present study revealed that 103 out of 139
msp-1 amplicon (74%) were monoclonal infections, whereas the remaining 36 (26%) was poly-allelic type, with 15% for (MAD20 + K-1), 5.7% for (MAD20 + RO33), 2.8% for (K-1 + RO33), and 2.1% were MAD20 + K-1 + RO33 type. The proportion of monoclonal infection was 48% MAD20, 13% K-1 and 13% RO33 (Table
2). This finding differ from the report from southwestern Ethiopia [
23,
34], where they reported that K-1 was the most prevalent allelic family. Similarly, report from Cameroon, Gambia, Nigeria and Gabon has shown that MAD20 allelic variant was the least predominant [
37,
38]. On the other hand, in agreement with the present study report from northwestern part of Ethiopia [
6], Sudan by [
7] and Equatorial Guinea [
22] of the three
msp-1 gene allelic families MAD20 was the predominant allelic type. Although the deriving forces for such variation needs further investigation; the difference in micro-ecological factors and the local transmission intensity [
39,
40], could play a significant role. Moreover, evolutionary process like genetic drift resulting uneven reproduction of the parasite lineages, types and rate of mutations, inbreeding and the contribution of allelic variants in reproductive success are some of the factors that might have contributed for such variation [
41]. In addition, in the present study when the spatial feature of the distribution of
msp-1 gene allelic variant in urban and rural areas (Table
4) was examined, no statistically significant (P = 0.2) variation was revealed. This finding could be taken as an evidence to show similar malaria epidemiology and the possible crossbreeding of the parasite populations between urban and rural settings in the study area, demanding similar intervention endeavours. Similarly in the present study, no statistically significant variation of multi-clonal infection of
msp-1 gene with parasite density (P = 0.6), and seasonality of transmission (P = 0.8). This could be due to the characteristic feature of low transmission settings in such malaria endemic regions [
42,
43]. On the other hand, study sites based distribution of allelic variants has shown a highly significant variation (P = 0.000), (Fig.
4). This could be due to the difference in local micro-ecology of the areas, intensity of local transmission pattern, and differences in the age of the study population [
36,
44] and the relative potential differences and challenges on the ongoing malaria control and elimination endeavours in those sites.
This study is the first attempt to analyse the most polymorphic gene (msp-1) of P. falciparum population in the study area. However, further characterization of this gene needs to be designed by increasing the sample size, use of the most powerful techniques, such as microsatellite DNA sequencing and capillary electrophoresis that would provide strong molecular evidence for malaria parasite genetic profile.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.