Background
Severe malaria is a major public health problem. Globally about 3.3 billion people live in malaria endemic areas, despite improvements in the implementation of effective control measures. Each year, there are an estimated 219 million clinical malaria cases and 435,000 deaths worldwide, the majority of which occur in sub-Saharan Africa [
1]. Approximately 60% of the Ethiopian population live in malaria-endemic areas [
2,
3].
Of the
Plasmodium species that cause human malaria,
Plasmodium falciparum is the main cause of severe malaria and malaria-associated deaths [
4]. Severe malaria is differentiated from uncomplicated malaria by a set of diagnostic criteria including clinical and laboratory criteria [
5]. The infective load is greater with
P. falciparum than other malaria species and its propensity to sequester in capillaries leads to increased morbidity [
6,
7]. However, the host-parasite factors leading to severe disease or death as compared to uncomplicated disease are not completely understood; these include parasite factors such as genetic diversity and multiplicity of infection (MOI).
Numerous studies have characterized strains of
P. falciparum isolates using genetic polymorphisms as markers [
8‐
11]. Merozoite surface protein (
msp) genes are the most commonly used markers of
P. falciparum due to their high polymorphism. MSP-1 and MSP-2 are
P. falciparum blood-stage malaria vaccine targets [
12] and are also suitable markers for identification of genetically distinct
P. falciparum parasite sub-populations. MSP-1 is major surface protein encoded by
msp-
1 on chromosome 9, which contains 17 blocks of sequences flanked by conserved regions. It plays a major role in erythrocyte invasion [
13] and is targeted by immune responses [
14]. Block 2, which is the most polymorphic part of
msp-1, is grouped under three allelic families of K1, MAD20 and RO33 [
15]. MSP-2 is a glycoprotein encoded by the
msp-
2 gene located on chromosome 2 and is composed of five blocks of which the central block is the most polymorphic. The
msp-
2 block 3 alleles are grouped into two allelic families, FC27 and IC3D7 [
16]. Genotyping polymorphic regions of
P. falciparum are important to determine diversity and multiplicity of
P. falciparum infection [
17]. Genetic diversity of
P. falciparum populations and MOI vary according to the intensity of transmission, outcome of infections and age in different geographical regions [
18]. In areas of high malaria transmission, parasite diversity and MOI are increased [
4,
18].
Some specific
P. falciparum genotypes have been associated with severe malaria in epidemiological studies [
19]. Severe malaria has also been associated with highly polymorphic parasites [
10] and multiclonal parasites [
11]. In comparison, other studies have observed a lower frequency of multiclonal infections in
P. falciparum isolates from patients with severe malaria [
20,
21]. Only a few studies have reported on the genetic diversity and clonality of
P. falciparum in Ethiopia [
22‐
24]. These studies have investigated isolates from uncomplicated symptomatic malaria. Thus, investigation of the
msp-
1 and
msp-
2 genes from isolates of patients with severe malaria in Ethiopia is important to obtain knowledge about parasite-factors associated with virulence in Ethiopia. This study aims to explore whether
P. falciparum genetic diversity and multiclonality are associated with disease severity and age of patients in Gublack area, northwest Ethiopia.
Discussion
Investigating the association between
P. falciparum genetic profiles and clinical outcome potentially provides important information for predicting disease-related outcomes in malaria endemic areas. This is the first study that has investigated the association between disease severity, and genetic diversity and multiplicity of infection using the two most polymorphic regions of
msp-
1 and
msp-
2 in northwestern Ethiopia. The
msp genes are usually used for
P. falciparum population genetics in spite of limitations of the impact of human immune selection [
32]. The findings suggest that malaria transmission is high in the study area despite efforts of intensive control measures. The authors also identified a greater propensity for multiclonal infections in patients with severe disease.
Associations between the dominant allelic families and disease severity were examined. In
msp-1, the K1 allelic family was identified in similar proportions in uncomplicated and severe malaria. This differs to previous studies in Senegal where the K1 allelic family had been associated with severe malaria [
33]. This difference may be due to differences in malaria endemicity or the small size of the current study, particularly in the severe malaria cases. The prevalence of two
msp-
2 families were also similar between uncomplicated and severe disease, which is similar to the previous reports elsewhere [
34,
35].
The study identified a higher proportion of
msp-
2 than
msp-
1, which is similar to a study conducted in Sudan [
9]. The current study found a high genetic diversity in parasites circulating in the study area with a total of 19 and 22 genotypes for
msp-
1 and
msp-
2, respectively. These findings are similar to those study reported from Senegal [
36]. The presence of more genetic diversity in the current study area is likely to be an outcome of the presence of more parasite population, that resulted in mixing of genotypes.
The current study also found a high frequency (64%) of patients with multiclonal infections, which is in line with the previous studies from Ethiopia [
22] and Sudan [
37]. In contrast, studies from West Africa including the Republic of Congo [
38] and Nigeria [
39], found a lower frequency of patients with multiclonal infections. This variation likely reflects differences in geographic variability, parasite epidemiology and transmission. The frequency of multiclonal infections has also been reported to increase with age until late childhood before declining [
40]. The results were consistent with this finding, with multiclonal infections more prevalent in younger children.
The mean number of circulating genotypes in severe malaria patients was higher than from patients with uncomplicated malaria. Similar results have been observed among patients with severe malaria in Uganda [
8]. Studies from Madagascar, Gabon and Sudan [
20,
34,
35] showed no differences in uncomplicated or severe malaria cases. However, a study from Nigeria reported a low a multiplicity of
P. falciparum infection in individuals with severe malaria [
41]. These differences may also be related to the immune status of the study population, considerable immigrant labourers were observed in the study area. Moreover, this could also be due to the differences in genotyping methods and interpretation of result, and the heterogeneity of the study populations may suggest the inconsistency variability of results between studies.
Age is a key factor involved in the acquisition of immunity against falciparum malaria and has been found to influence MOI [
8]. In turn, the low levels of acquired immunity in young children may be a major factor contributing to their vulnerability to control the infection [
42,
43]. Although the mean MOI decrease with age was observed in the current study this was not statistically significant. These results complements the findings from Ethiopia [
22], Republic of Congo [
38], Senegal [
44], and south of Benin [
45], but contrast with a study from Tanzania [
43]. The lack of a significant trend is potentially limited by power due to the relatively small number of patients younger than 15 years. The high level of malaria transmission in the region may be expected to lead to a higher risk of severe malaria in younger patients where immunity is lower [
9], however, the mean age of the patients with uncomplicated and severe malaria was similar.
Bed nets are an important tool for prevention of the malaria vector and are widely used in malaria endemic areas of the Ethiopia. Reduced MOI has observed to be associated with increased ITN use, which is consistent with being an indicator of transmission intensity [
46]. The present study found a low utilization of bed nets (24.6%), which might be due to the location of the study area, where there are mechanized farms with migrant labourers sleeping in open fields or temporary shelters [
25]. This poor uptake of bed nets might be an important factor contributing to the increased the MOI, with labourers at high risk of developing malaria infection in this area.
Consistent with previous studies, the current study found no association between the frequency of specific
P. falciparum alleles and disease severity [
12,
35]. Limitations in the current study include the small number of patients with severe disease and the use of nested PCR instead of microsatellites or DNA sequencing which could potentially underestimate genetic diversity. Furthermore, with the rapid temporal changes in parasite density and maturity due to parasite sequestration, a single peripheral blood sample may not reveal the full complexity of the parasite population harbored by individuals [
47]. In addition, low frequency alleles at the time of blood sampling may not have been detected by nested PCR. Future studies need to be designed to take large sample sizes and use more robust techniques to study the relationship between genetic diversity and malaria severity.
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