Genetic polymorphisms of Plasmodium falciparum isolates from Melka-Werer, North East Ethiopia based on the merozoite surface protein-2 (msp-2) gene as a molecular marker

The study samples were collected from Melka-Werer rural area in Afar Regional state in North East Ethiopia, a sentinel site for monitoring of anti-malarial therapeutic efficacy (Fig. 1). The study area is located 291 km northeast of Addis Ababa at an altitude of 723 m above sea level. Melka-Werer is one of the kebeles of Amibara district, with a catchment population 61,222 inhabitants; the majority living as pastoralists or semi-pastoralists [22]. The semi-arid climatic zone has a long hot summer, and a short mild winter with annual rainfall between 200 and 500 mm. Malaria transmission is markedly seasonal, with a peak during August to December. Plasmodium falciparum and P. vivax are the two dominant malaria parasites in the region. The local vector responsible for most malaria transmission is Anopheles arabiensis with mosquito breeding predominantly occurring adjacent to the Awash River and malaria distribution reflecting this. The prevalence of malaria in this region is declining according to Ethiopian national malaria indicator surveys; with a cross-sectional prevalence of 2.4% in 2007, 0.8% in 2011 and 0.2% in 2015 [6, 23, 24].

Dried blood spot samples were collected from children and adults presenting to the Melka-Werer Health Centre with P. falciparum malaria and enrolled into a 28-day therapeutic efficacy of artemether-lumefantrine which found 100% adequate clinical and parasitological responses. The study was conducted from September 2014 to January 2015. Individuals with an axillary temperature of  ≥ 37.5 °C or history of fever within the previous 24 h, haemoglobin (Hb) level > 5 g/dL, microscopically confirmed P. falciparum mono-infection with asexual parasite densities between 1,000 and 100,000 parasites/µL blood and residence within the study area were eligible for enrolment in the study [25]. Thick and thin blood films were stained with 3% Giemsa for 45 min and slides were read by two health centre laboratory technicians. In case of discordant results, a third WHO-certified microscopist resolved the discrepancy. Using the thick blood film, asexual parasitaemia was counted against 200 leucocytes and expressed as the number of asexual parasites/μL of blood, assuming a leucocyte count of 8,000/μL of blood [26]. Haemoglobin was measured from finger prick blood samples using a portable spectrophotometer (HemoCue Ängelholm Sweden). Before treatment, approximately 50 µL of blood from each patient was spotted onto Whatman 903® filter paper (Schleicher & Schuell BioScience), air-dried and stored in labelled zip lock bags with desiccant before being transported and stored at − 20 °C from 2015 to 2020 in the Malaria Research Laboratory at the Ethiopian Public Health Institute (Fig. 2).

DNA was extracted from the dried blood spots using the Chelex-Saponin method [27] and allelic family-specific analyses of msp2 block 3 were carried out as previously described [20]. The allele- specific positive control 3D7 and DNA free negative controls were included in each set of reactions [16]. The nested PCR products were separated by electrophoresis on 2% agarose gel in 1X TBE (Tris borate EDTA) buffer stained with 0.5% (v/v) ethidium bromide at 80 V for 30 min. After running the gel, it was placed in a gel documentation system (Cleaver Scientific UV Transilluminator, UK) that was connected to a desktop computer to visualize the bands under ultraviolet transillumination. The size of DNA fragments was estimated visually based on their mobility related to a 100 bp DNA ladder marker (Boehringer Mannheim Marker VI). Alleles in each family were considered the same if fragment size was within a 20 bp interval [28]. The MOI was calculated by dividing the total number of distinct msp2 fragments observed by the number of positive samples. Isolates with a single genotype were considered monoclonal infections and those with more than one genotype as polyclonal infections. The frequency of monoclonal infections was the number of patients with one parasite genotype divided by the total infected population. Heterozygosity index was calculated using the following formula He = n/(n−1) (1- ΣPi²), where n is the number of isolates sampled and Pi is the allele frequency [29].

Data were recorded in Excel and analysed with SPSS version 20 (SPSS Inc., Chicago, IL, USA). The frequency of msp2 allelic families was calculated as a proportion of all detected alleles in the isolates. Spearman’s rank correlation coefficient was calculated to assess the association between MOI and mean parasite density and age. The non-parametric Mann–Whitney U test was used to compare the association between MOI and previous exposure to malaria. A P value < 0.05 was considered statistically significant.

A total of 72 patients were confirmed to have malaria. Of these, 52 (72%) patients were confirmed to have P. falciparum malaria and were genotyped for the msp2 alleles. An equal proportion of males (26, 50%) and females (26, 50%) were enrolled. The patients’ ages ranged from two to 60 years (mean 21.3 ± 14.4). The geometric mean parasitaemia was 3,661 (95% CI 2,768–4,877) ranging from 1,000 to 83,200 parasites/µL (Table 1). The mean axillary temperature was 38.1 °C (95% CI 37.8–38.4). 90.4% of the study participants could not recall previous exposure to malaria. The geometric mean parasite density of individuals with previous exposure to malaria attack was higher 10,854 parasites/µL compared with 3,226 parasites/µL in those without prior exposure.

All samples positive for P. falciparum were genotyped for msp2 Block-3 by nested PCR. msp2 was identified in 41 (78.8%) samples. A total of fourteen different alleles were identified in msp2 genotyping. Six alleles of 3D7/IC (280–500 bp) and 8 alleles of FC27 (180–450 bp) were detected. The distribution of the corresponding band size is presented in Additional file 1: Figure S1. The proportion of isolates with only 3D7/IC and FC27 alleles was 34.1% (14/41) and 22.0% (9/41), respectively. Both msp2 allelic families were identified in 43.9% (18/41) of the isolates (Table 2). 40.4% (21/52) of the isolates contained multiple msp2 alleles and the overall mean MOI was 1.2 (95% CI 1.00–1.40). The heterozygosity index for the msp2 locus was 0.5.

A negative correlation was observed between parasite density and MOI (Spearman rank coefficient = − 0.33 P = 0.022). Age was not correlated with the MOI (Spearman rank coefficient = 0.080; P = 0.51). The highest MOI was in the 10–18-year-old age group as shown in Table 3. There was no association between the MOI of individuals with previous exposure to malaria attack compared to those without previous exposure (p = 0.935).