Background
Malaria, a disease mostly caused by
Plasmodium falciparum, is a major public health problem. The global burden is 207 million malaria cases every year resulting into 627,000 deaths [
1], sub-Saharan Africa being the most affected region. In Ethiopia, malaria still remains a major health problem with about two-thirds of the population living in malarious areas [
2]. Among the malaria parasites,
P. falciparum is the most fatal species, and exhibits complex genetic polymorphism which may explain its ability to develop multiple drug resistance and circumvent vaccines [
3].
Recent, studies of anti-malarial drug resistance in Ethiopia have confirmed high level of resistance to sulphadoxine-pyrimethamine. Consequently, in 2004, Ethiopia changed its anti-malarial drug policy to artemisinin-based combination therapy (ACT) Coartem® as first-line drug for the treatment of uncomplicated
falciparum malaria [
4,
5]. Following this, in 2005, the country launched a scaling up of its control and prevention programme with strategies that lead towards malaria elimination in the country [
6]. However, since, there is lack of pre-elimination data of malaria parasite population circulating in the country, it was found important to study the genetic diversity, using the two highly polymorphic markers, merozoite surface protein 1 and merozoite surface protein 2.
Merozoite surface protein 1 and 2 of
P. falciparum are major blood-stage malaria vaccine targets [
7] and are also suitable markers for the identification of genetically distinct
P. falciparum parasite sub-populations. MSP1 is major surface protein of approximately 190-kDa size. It plays a major role in erythrocyte invasion [
8] and is a major target of immune responses [
9]. MSP1 contains 17 blocks of sequence flanked by conserved regions [
10] Block 2, which is the most polymorphic part of MSP1, is grouped into three allelic families namely K1, MDA20, and RO33 type [
11]. MSP2 is glycoprotein consisting five blocks where the central block is the most polymorphic [
12]. MSP2 alleles are grouped into two allelic families, FC27 and 3D7/IC1.
Based on the literature, there is no published data available on the diversity and multiplicity of infection in uncomplicated falciparum malaria in the country. Such data have importance in documenting the parasite genetic diversity changes due to elimination pressure through the scaled up malaria control intervention. This study was under taken to collect background data on the extent genetic diversity of P. falciparum in the pre-elimination period from Kolla-Shele in southwestern Ethiopia.
Discussion
In Ethiopia, less attention has been given to investigating the genetic diversity of P. falciparum than other countries. This is the first MSP based study to provide information about the genetic diversity of P. falciparum in Ethiopia. These finding may be an important element for implementing malaria control strategies in the country, as elimination may influence genetic diversity and their heterozygosity.
The allele specific
P. falciparum msp1 and
msp2 genotyping has shown that malaria parasite population in Kolla-Shele area is moderate to high allelic diversity. However, the number of alleles may have been underestimated due to the limitation of the technique. Indeed, fragments with the length interval of less than 20 bp could not clearly be distinguished as a separate allele. In their allelic frequency, out of the 59 allelic types detected in
msp1, the K1 allelic family with 33.9% allelic frequency besides its representation in poly allelic bands, was found predominant This is in line with previous studies in Central Africa, Gabon, Benin, and Ghana [
18,
22-
24], but in contrast to studies from Sudan and Malaysia where RO33 allele was predominant [
25,
26]. With regard to
msp2, 39 allelic types were found and the alleles belonging to 3D7/IC1 family were more frequently detected (21.5%). This is similar to data reported from Kenya, Congo Brazzaville and other sub-Saharan-African countries [
10,
20,
27], as well as Peru and Iran [
28,
29].
The entomologic inoculation rate (EIR) study conducted in the same region showed a value of 17.1 infectious bites per person per year [
14], the moderate to high EIR value reported shows the meso-endemicity of malaria transmission which is in agreement with this finding of moderately high genetic polymorphism of 60% and MOI of 1.8. It is reported that the multiplicity of infection in an infected patient may be due to an important entomological inoculation rate as shown in the Senegal where patients are exposed to a large number of infective bites [
30].
The Nei unbiased heterozygosity index (H
e) was high for
msp1 (0.79), indicating a larger genotype diversity within the
msp1 locus than that of
msp2, with H
e 0.54, which is nearly 0.5, i.e. the maximum expected Nei diversity index for allelic locus. This is almost parallel to the Hardy Weinberg’s 0.5 “2pq” equilibrium heterozygosity, in equal frequency P and q alleles. Similarly a moderate heterozygosity level of 0.51 < He 1998-1999-2002 < 0.58 was observed in the population diversity of
P. falciparum in Djibouti [
31]. This, however, indicates only the state of equilibrium heterozygosity at the microsatellite loci, which by 2009 reached a state of total similarity, He = 0, indicating the monoclonality of the Djibouti strain at the studied loci. What will be found in the future with the process of elimination peaking pace in Ethiopia remains to be observed, especially with the highly divergent allelic locus of
msp1.
The predominant families in the
msp-1 and
msp-2 were K1 and 3D7/IC1, respectively, indicating that both K1 and 3D7/IC1 are good indicators for determination of MOI, at least in Southern Ethiopia. This is because high numbers of bands were encountered with genotypes of these two allelic families. These findings are in agreement with previous study in Republic of Congo [
20]. But it is in contrast to studies in Uganda and the Sudan [
25,
32] where RO33and FC27 allelic families were found predominant.
The FC27 fragment of 400 bp, which was the most prevalent in clinical episodes of malaria among symptomatic malaria children, similar observations were reported from the Republic of Congo and Burkina Faso [
33,
34]. This may indicate an association between this FC27 allelic type and clinical episode.
This study also showed that the number of parasite genotypes carried by subjects with symptomatic infections was not influenced by age. This is in agreement with previous reports in Republic of Congo and Gabon [
20,
22]. This may be besides PCR limitations, possibly other factors like comparable level of immune naivety of young and older children in the sample population not to selectively “weed out” certain circulating genotypes.
The MOI values reported in this study was higher than found in countries like Malaysia where the multiplicity of
P. falciparum infection was 1.37 and 1.20 for
msp1 and
msp2, respectively [
26]. but lower from findings in Côte d’Ivoire, where their MOI was found to be 2.88
msp2 [
35]. This discrepancy may be due to differences in geographical areas and their transmission patterns (differing malaria endemicity level) and also due to differences in sample population determination. Generally, however, the higher the malaria transmission level, the greater becomes the tendency to get a higher MOI and mean number of alleles per locus. Thus in neighbouring Djibouti,
P. falciparum genetic diversity study showed a decrease of MOI from 1.42 at the peak transmission year in 1999 to 1.12 in 2002 and just 1.0, in 2009, the time the control programme advanced in its pre-elimination phase. With 1.8 MOI, this is nearly two alleles per locus and parallel to this polyclonality, as explained above is observed a moderate (
msp2) to high (
msp1) H
e values.
The present study found that about two-third of the samples (59%) harboured multiple genotypes; almost similar frequency (62%) to that in the Sudan [
25], while 83% of the sample population harboured multiple genotypes
, in the Republic of Congo [
20]. This may go with unstable seasonality of transmission in most malarious areas of Ethiopia, which is more similar with the Sudan than that of West Africa.
The mean MOI of persons with previous exposure to malaria attack is higher compared to persons with absence of previous malaria attack (non-exposed) i.e. high frequency of MOI correlates with high frequency of parasitic density. The finding may indicate that persons with lower parasitic density may have low acquired immunity (higher risk of clinical malaria), that they become symptomatic at a lower parasite threshold, unlike those with previous exposures. This is a tacit indicator that just few previous exposures, can elicit certain level of clinical immunity to make them tolerant to lower parasitaemic threshold up to 10,000Ps/ul blood.
This study represents a first attempt to analyse the molecular characteristic of P. falciparum population. However, future study needs to be designed to increase the representative sample sizes in different transmission areas and use more robust techniques, such as microsatellite DNA sequencing, to study in depth the molecular diversity of the P. falciparum parasite.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HM was fully involved in all phases of the study, including in laboratory during Molecular analysis, data analysis, interpretation, and write-up of the manuscript; TM and MB were designed the study project critical revised the manuscript. MT was involved in statistical analysis of data and critical revision of the manuscript for publication; TM MK, AA, AW and AM were contributed to write up. All authors read and approved the final manuscript.