Background
The intensification of malaria control interventions has resulted in its global decline, but it remains a significant public health burden across several malaria-endemic countries [
1]. The 2018 global malaria report revealed that the incidence rate of malaria declined by 18% from 2010 to 2017, in the same period, the estimated number of cases dropped from 239 million to 219 million, and the number of deaths from 607,000 to 435,000 [
1‐
3]. In Ethiopia, the trends in malaria over the past five years have also shown a decline in malaria cases and fewer epidemics [
4,
6]. In 2014/2015, Ethiopia reported 2174,707 malaria cases and 662 reported malaria deaths among all age groups which is a 98% reduction compared to 41,000 estimated deaths in 2006 [
4,
6]. Between June 2016 and July 2017, the Ethiopian Health Management Information System (HMIS) reported a total of 1,755,748 malaria cases and 356 deaths due to malaria [
4]. The key interventions which have been contributing to such significant decline includes: introduction of prompt and effective treatment with artemisinin-based combination therapy (ACT), the distribution and promotion of the use of long-lasting insecticidal nets (LLINs), nationwide coverage of indoor residual spraying (IRS), and environmental management [
4‐
6]. Ethiopia adopted artemether-lumefantrine (AL) in 2004 as first-line for the treatment of uncomplicated falciparum malaria, LLINs coverage has been scaled up in Ethiopia since 2005, resulting in over 64 million nets distributed by 2014. IRS, including permethrin, bendiocarb propoxur and deltamethrin, pirimiphos-methyl has been used between 2014–2020. Although these control measures have resulted in a substantial decrease in malaria infections in Ethiopia, malaria is still endemic, with populations in some areas remaining at high risk of infection. Ethiopia has set a goal to eliminate the disease by 2030 using these interventions [
4,
7].
Genetically-distinct malaria parasites in natural populations have an extremely high rate of genetic recombination during the sexual stages in a mosquito host, often resulting in multiple strains being transmitted simultaneously [
8]. This diversity hampers development of effective vaccine as it limits the efficacy of protective immunity (i.e., antibody–mediated parasite inhibition) [
9]. Highly endemic malaria settings are prone to infections containing multiple
P. falciparum strains, primarily due to repeated exposure to mosquitoes infected with multiple parasite strains [
10]. This genetic diversity of the parasite is one of the main factors responsible for the slow acquisition (several years) of immunity against malaria. Thus, individuals would have to encounter a broad range of circulating parasite populations before they develop an effective anti-malarial immunity [
11].
Genetic diversity and multiplicity of
P. falciparum infections are essential parasite indices that could determine the potential impact on the selection of drug-resistant parasites. Although many polymorphic antigens have been described in several stages of the parasite life cycle, merozoite surface protein 1 and 2 (
msp-
1 and
msp-
2) seem to be the most appropriate to distinguish parasite populations [
12‐
14]. This markers are particularly useful in determining the multiplicity of infection (MOI), a measure of the effectiveness of intervention programmes and also
msp-
1 and
msp-
2 typing are widely used in anti-malarial drug efficacy trials to distinguishing recrudescent parasites from new infections [
15‐
17]. Study reports by Jelinek et al. [
18] and Meyer et al. [
19] showed that increased genetic diversity of circulating malaria parasites in a population in-creases the potential for the selection of drug resistance.
Declining malaria transmission as a result of scaling-up interventions has been shown to affect the parasite population genetics pattern and population structure of
P. falciparum [
20‐
22]. The scale-up interventions, such as the usage of insecticide-treated bed nets, indoor residual spraying [
21,
23] and the introduction of new anti-malarial drug regimens [
20,
24‐
29] to control and treat malaria have been shown to cause the genetic drift and decrease the level of allelic diversity(
He) and MOI. However, this does not occurred in all settings [
20,
21]. In addition, the genetic diversity and population structure studies can be used to monitor the effects of any malaria scale-up interventions, such as the impact of malaria control and elimination programs [
30]. Hence, accurate assessment of the parasite’s genetic diversity across malaria endemic regions could help plan or develop new control and elimination strategies. The MOI, which identifies the number of clones within a particular infection, can serve as a measure of the level of malaria transmission as well as identify hotspots [
31,
32]. Malaria parasite diversity is distinct in different individuals, populations, transmission settings and seasons within endemic zones and changes with variations in parasite prevalence [
33], and has been suggested to be constantly changing [
34‐
37]. Parasite populations even respond to specific interventions, such as rapid diagnostic tests, human host immune pressure and mosquito vector [
38‐
40]. The identification of hotspots is important in understanding the epidemiology of
P. falciparum infections for informed interventions to be implemented [
32,
41]. The effect of malaria control interventions on the
P. falciparum population structure in Ethiopia could not be assessed due to the lack of genetic data and systematic genetic surveillance study. Chewaka district in Southwest Ethiopia experiences frequent epidemic outbreaks of malaria. Parasite genetic diversity and multiplicity of infection studies have also been found to be important in the surveillance of strains circulating in a particular transmission area especially in Southwest Ethiopia because there was so limited information available on the genetic structures of
P. falciparum [
42‐
44]. This study was aimed at characterizing the genetic diversity and allele frequencies of
msp-
1 and
msp-
2 genes of
P. falciparum isolates from uncomplicated malaria patients in Chewaka district, Southwest Ethiopia.
Discussion
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 [
14] 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 (160 bp). 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 [
56] and Central Sudan [
14], 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 [
57‐
60]. 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 and 2018 [
42‐
44], Brazzaville, Republic of Congo (MOI: 2.2) [
16] in 2011 and Bobo-Dioulasso, Burkina Faso (MOI: 1.95) [
61]. In contrast to study reported in Bioko Island, Equatorial Guinea (MOI: 5.51) [
53] in 2018 and Gabon (MOI: 4.0) [
62] 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 [
14]. 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 [
56‐
59]. 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 [
63]. Studies have shown an age-dependent MOI in a village with intense perennial malaria transmission but not in areas where malaria is mesoendemic [
50,
58]. 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 [
56]. 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 [
57,
64,
65]. Besides, transmission intensity can also be affected by other factors, such as vector biting behaviour and endemicity [
64]. 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 [
64]. 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.
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