An Evolution From a Predominant K1 Allelic Variant to MAD20 of msp1 Gene Between 2015 to 2019 in Metehara, Southeast Ethiopia

Abeba Reda (  abebagtsadik@yahoo.com ) Ethiopian Public Health Institute Alebachew Messele Addis Ababa University, Aklilu Lemma Institute of Pathobiology Hussein Mohammed Ethiopian Public Health Institute Ashena Assefa Ethiopian Public Health Institute Lemu Golassa Addis Ababa University, Aklilu Lemma Institute of Pathobiology. Hassen Mamo Addis Ababa University, department of microbial, cellular and molecular biology.


Background
Malaria is one of the worst infections, claiming the lives of about 400,000 people worldwide. Despite its tremendous reduction in the previous two decades, malaria remains one of Ethiopia's biggest public health and socioeconomic challenges [1,2]. Around 68 percent of Ethiopia's population lives in malaria-endemic areas [3]. Ethiopia has achieved a signi cant reduction in malaria cases and has entered the pre-elimination phase across the country. A change from high or moderate to low malaria transmission necessitates a better understanding of the current parasite population's distribution, dynamics and genetic structure, all of which are critical for achieving and maintaining eradication [2,15].
Genotyping of a polymorphic region such as block 2 of the merozoite surface protein 1 (MSP1), block 3 of the merozoite surface protein 2 (MSP2) or the RII repeat region of the glutamate rich protein (GLURP) is generally used to determine P. falciparum genetic diversity [6][7][8]. The three polymorphic genes (msp1, msp2, and glurp) have been extensively researched [9,11,18,20,26] and are valuable for determining genetic diversity and infection multiplicity. P. falciparum merozoite surface proteins 1 and 2 are important targets for blood-stage malaria vaccines [7] and can also be used to identify genetically diverse P. falciparum parasite sub-populations. MSP1 is a 190-kDa main surface protein that plays a key role in erythrocyte invasion [7,8] as well as being a primary target of immune responses [6]. msp1 is divided into three allelic families: K1, MDA20 and RO33 type [14]. Block 2 comprises 17 sequence blocks that are separated by conserved portions. MSP2 on the other hand, is a ve-block glycoprotein with the middle block being the most polymorphic. Allelic groups FC27 and 3D7/IC1 both contain msp2 alleles. Glurp is a 220 kDa antigen that is produced throughout the malaria parasite's life cycle [6,22,31].
The collection of baseline data for these polymorphic biomarkers in the parasite population from various geographical regions and levels of malaria transmission is critical for current malaria control efforts in Sub-Saharan Africa, particularly Ethiopia [10,11,26]. As a result, the current study sought to investigate the genetic diversity of msp1, msp2 and glurp in natural P. falciparum populations in Metehara in order to gain a better understanding of the temporal changes in the diversity of these polymorphic markers following the implementation of massive intervention strategies in Ethiopia between 2015 and 2019.

Study site
Samples used for this study were collected from Metehara, south east Ethiopia, a sentinel site for monitoring of therapeutic e cacy to artemether-lumefantrine (Coartem®) in Ethiopia. (Fig. 1 PCR amplifcation and allele detection of msp1 gene Genomic DNA was ampli ed using allelic speci c primers by nested PCR (Additional les 1): (supplementary Table  1) Table S1. PCR ampli cation procedures were followed as previously described [13]. A thermal cycler was used to heat all of the PCR reaction mixtures (Perkin-Elmer Cetus PE 9600, Bio-Rad, Hercules, USA).
In a nal volume of 20µl PCR reactions were carried out. 4µl gDNA, 10 µl GoTaq Green Master Mix (Promega), 0.5µl (0.5 M) of each primer, and 5µl nuclease free water were used in the primary round reaction. The secondary reaction was identical to the rst reaction with the exception of 2µl of PCR amplicon.
Initial denaturation at 95°C for 3 min was followed by 35 cycles for primary and 30 cycles for secondary reactions of denaturation at 95°C for 1 min, annealing at 58°C for 2 min, and extension at 72°C for 1:30 min, with a nal extension at 72°C for 5 min. Each set of reactions comprised positives (3D7) and DNA-free water as negative controls.
Genotyping of msp2: Except for the family-speci c primers, PCR reactions and master mix preparation were carried out similarly to msp1. The primers used to genotype the polymorphism areas of the msp2 gene in P. falciparum isolates are mentioned in: (Additional File 2) (Supplementary Table 2) Table S2.
Genotyping of glurp PCR reactions were carried out in a nal volume of 20 µl containing: 5 µl gDNA, 7.5 µl GoTaq Green Master Mix (Promega), 0.5 µl (0.5 M) of each primer, and 6.5 µl nuclease free water in initial rounds. In the secondary reaction, 2µl of PCR amplicon product was mixed with 9.5µl nuclease-free water in a 7.5µl GoTaq Green Master Mix and 0.5 µl (0.5 M) of each primer. The following were the cycling conditions for the primary and secondary PCR reactions: For the primary glurp PCR, the following conditions were used: 95°C for 3 minutes, 94°C for 1 minute, 45°C for 1 minute, 68°C for 3 minutes, 72°C for 3 minutes, followed by 30 cycles. Secondary PCR process at 94°C for 1 minute, 55°C for 2 minutes, 70°C for 2 minutes and 72°C for 3 minutes.
PCR products were resolved in 2 percent agarose gels (Caisson, Utah, USA), stained with ethidium bromide submerged in 0.5 TBE (Tris-borate EDTA) buffer electrophoresis at 120 volts, 400 ampere for 45 minutes, and visualized under UV trans-illumination and photographed at 302 nm on a gel documentation system (VersaDoc®, Bio-Rad, Hercules, USA). A 100 base pair (bp) DNA ladder marker was used to visually evaluate the size of DNA pieces (New England Biolabs. Inc, UK). A polyclonal infection was de ned as the presence of more than one genotype, whereas a monoclonal infection was de ned as the presence of only one allele. For the quality control of alleles in each family, fragment sizes were calculated within a 20-bp interval for merozoite surface protein 1,2 and a 50-bp interval for glurp [25]. During the PCR cycle, both a positive control and a negative control were performed with the test for quality control purposes. The alleles were identi ed by comparing them to their genomic controls.

Ethical clearance
Ethical approval of the study was obtained from Institutional Ethical Review Board of Addis Ababa University (AAU), certi cate reference number IRB/033/2018. In addition, written informed consent and/or assent were obtained from the parents and guardians of children and malaria positive individuals were treated according to national malaria guidelines in the health center [12].

Data analysis
The program IBM SPSS version 20 was used to conduct all statistical analyses (SPSS Inc. Chicago, IL, USA). The allelic frequency and mean MOI of the msp1, msp2 and glurp genes were calculated using proportions of allele comparisons and Chi square tests. The MOI was compared using the student t test between 2015 and 2019. To assess the relationship between MOI, parasite densities and patient age groups the spearman's rank correlation coe cient was calculated. P<0.05 was selected as a threshold for statistically signi cant differences. The expected heterogeneity (He) was calculated by the formula; He = n n − 1 1 − ∑ p 2 where "n" stands for the number of the isolates analyzed and "p" represents the frequency of each different allele at a locus [14].

Results
Demographic and parasitological data Microscopy, Rapid diagnostic test and quantitative PCR (qPCR) were used to diagnose a total of 83 screened from 2015 (N=33) and 2019 (N=50). (Additional les 2): (supplementary Table 2) Table S2. The study population's demographic and clinical characteristics are shown in (Table 1) Microscopy and RDT together analysis identi ed 81.9%. QPCR analysis did identify 84.3%. One case of mixed P. falciparum/ P. vivax infections. This one misdiagnosed sample was not included in the genotyping ((Additional les 2): (supplementary Table 2) Table S2.

Discussion
Malaria transmission and management tactics have been impacted by the genetic variety of P. falciparum parasites. In low-endemic locations, it's critical to gure out the genetic population structure of P. falciparum parasite isolates. Using three polymorphic antigen markers, we compared the genetic diversity of P. falciparum populations in Metehara, Oromia Regional State, South East, Ethiopia, between 2015 and 2019. Genotyping the two msp markers msp1 and msp2 revealed much more allele variations than genotyping glurp [11,17,18,19], con rming previous ndings [11,17,18,19]. QPCR analysis did identify 84.3%. One case of mixed P. falciparum/ P. vivax infections. This one misdiagnosed sample was not included in the genotyping.
In the msp1 gene, K1 was the most prevalent allelic family in 2015, with 19 (65.5%) more than in 2019, in contrast to prior studies by others, in which Mad20 was the most prevalent allelic family 18 (43.9%) and this is comparable to what was observed in North West Ethiopia [39]. South West Ethiopia [28,40], Cameron [10], Senegal-Nigeria [30] and Madagascar [32] are among the countries represented. MOI calculations for each gene revealed that the mean MOI for msp1 was lower than msp2 and greater than glurp, which is consistent with the ndings of [39,10,21].
FC27 type was the more common allelic family in msp2 gene, which was similar to what had been reported from North West Ethiopia [21] However, differed from data from South West Ethiopia [28], North East Ethiopia [23], Burkina Faso [33] and Cameron, where IC/3D7 was the more common allelic family. These discrepancies could be due to the rift valley's geographical location and reduced transmission intensity compared to South East Ethiopia's hot and humid climate.
The glurp genotyping between 2015 and 2019 both had the 551-600 base pair allele as the most frequent, whereas the 501-550 bp alleles were the most common ones from 2015. Limited genetic diversity of P. falciparum was observed in this study. Similar results have been reported in other areas like Northeast Ethiopia and Djibouti neighbor countries with low P. falciparum transmission [23,29].
Expected heterozygote (He) diversity varied from 0.3 for msp1 in 2015 to 0.03 for msp1 in 2019, indicating that the parasite population in Metehara has intermediate to low heterozygosity, indicating decreased genetic diversity [29]. In our study low genetic diversity and high allelic frequencies seen a difference from previous study have been reported from other sites including South Western Nigeria [19], Burkina Faso [33], Cameron [10] and from Ethiopia The declining malaria transmission, as a result of scaling up interventions, has been shown to affect the genetic diversity pattern and population structure of P. falciparum [29,34]. Limited genetic diversity of P. falciparum was observed in this study. Similar results have been reported in other areas with low P. falciparum transmission [34] As a result, the low MOI found in this study implies low malaria transmission intensity in Metehara. This is consistent with previous studies, which linked an increase in MOI to increased malaria endemicity and a low MOI for msp1 and msp2 to low malaria transmission intensity [11,29,34]. Like contrast, high endemicity settings in Cameroon [10] and Burkina Faso [33] have been observed to have a high amount of genetic diversity. The current study found that the P. falciparum parasite population in Metehara overall exhibited a low heterozygosity 0.48, 0.70, 0.55 for msp1, msp2, glurp, respectively consistent with that reported in Northeast Ethiopia for msp2 (He=0.5) [33].
Heterozygosity in 2015 and in 2019 was 0.3 and 0.03 this is similar to the study report in East Asia Sabah which was He =0.33 (34).
In areas with declining local transmission, it is expected that lower parasite diversity (heterozygosity) will be present [34]. Declining diversity and transmission have been associated with improved malaria control interventions [29,33,34].
In this study, the total mean MOI (MOI=1.67) was low. Low malaria transmission areas are often associated with lower MOIs [33,35]. This is in line with reports from Sudan and Djibouti's semi-desert environments [36,29]. The majority of the research participants were over the age of 15, which is consistent with results from a malaria transmission area with lower intensity [33]. Age is thought to play a role in the development of P. falciparum immunity and may have an impact on MOI [38]. However, the most recent study found no link between age and MOI. Similar ndings have been found in other countries [39]. As a limitation of this study, allele distinction should be improved in future studies by using more selective techniques like DNA sequencing or SNPs.

Conclusion
The majority of P. falciparum infections in Metehara, Oromia Regional State, South East Ethiopia, were monoclonal, according to this study. This is consistent with the low infection rate in this area. Further research in similar low-transmission conditions with greater sample sizes is required. Further research into the dynamics of P. falciparum malaria variety in Ethiopian regions such as sequencing or SNPs is needed. In this study, there was an improvement in the reduction of MOI during a ve-year period. The development of the K1 variant by the MAD20 variant was also seen. As a result of the changing epidemiology of malaria, this study supports the use of the glurp, msp1, and msp2 genes in the characterization of Plasmodium falciparum infection, especially when the MOI is one of the primary parameters to be examined for malaria control strategies. Authors' contributions AG was fully involved in all phases of the study, including in laboratory during Molecular analysis, data analysis, interpretation, and write-up of the manuscript; HI and LG were designed the study project critical revised the manuscript. AL was involved in statistical analysis of data HM, AS, were contributed to Data collection and critical revised the manuscript. All authors read and approved the nal manuscript Funding No funding was obtained for this study.

Availability of data and materials
All relevant data is included in manuscript, and the datasets analyzed in the study is available from the corresponding author on reasonable request. Additional data uploaded with main document

Competing interests
We declare that we have no competing interests.

Ethics approval and consent to participate
The research and ethical committee of Addis Ababa University Institutional Review Board (IRB)reviewed and approved the study protocol, as veri ed through certi cate reference number IRB/033/2018 Addis Ababa University also approved the study protocol.   Figure 1 <p>Map of Metehara the study area, showing sample collection sites.</p> Figure 2 <p>Relationship between geometric mean parasite density and multiplicity of <em>P. falciparum infection</em> </p>