Study site
Samples used for this study were collected from Metehara, south east Ethiopia, a sentinel site for monitoring of therapeutic efficacy to artemether-lumefantrine (Coartem®) in Ethiopia. (Fig. 1).
Metehara is located 8°33′N 39°16′E / 8.55°N 39.27°E / 8.55; 39.27 at an elevation of 947m (3,107ft), 134 km southeast of Addis Ababa. Metehara is the largest town in Fentale woreda. This study site is one of the settings carefully selected by the National Malaria Control Program (NMCP) as sentinel site to carry out epidemiological survey in order to obtain information paramount in guiding the national malaria management policy. The study was carried out between November to December 2019 during the peak transmission season at the Metehara health center. Metehara is situated in the rift valley area. Awash river and basin have a suitable habitat to support mosquitoes that transmit malaria. The sugar factory estate irrigation system depends on the nearby the Awash River for the cultivation of sugar-cane. The Beska river, provides breeding sites for the Anopheles mosquito. Currently An. Stephani, a new vector has been seen in Metehra and susceptibility for this new vector also done to support malaria elimination in Ethiopia [16, 17]. Malaria transmission in this area occurs perennially with peaks during the two rainy seasons (September – November and March - May). This area is endemic for both P. falciparum and P. vivax.
Study design
A cross-sectional study conducted in Metehara from November to December between 2015 and 2019.
Study population and blood sample collection
Plasmodium falciparum clinical samples were collected in 2015 during efficacy studies of artemisinin combination therapies conducted in Metehara and in 2019 from outpatients who had a history of fever within the previous 24 hours and symptoms consistent with clinical malaria during their visit to Metehara Health Center. Malaria quick detection kit (CareStartTM malaria Pf/Pv Combo test Lot. No. G40IR, expiration date 2021 June,13, Access Bio, Inc., New Jersey, USA) was used to draw finger-prick blood samples and evaluated them. Microscopy, Rapid diagnostic test and quantitative PCR (qPCR) were used for the detection of infections
Dried blood spots (DBS) were collected for molecular analysis using Whatman 903® filter paper (Schleicher & Schuell Bio Science, Keene, NH 03431, USA). The DBS were transferred to Ethiopian Public Health Institute's Malaria Research Laboratory and stored at 20°C until they were evaluated.
Exclution createria: Patients critically ill and unable to give blood, patients who have other serious chronic disease were excluded.
Inclution createria: Patients who do not have serious chronic disease were included in this study.
Genomic DNA extraction
A tiny section of the blood-blotted cards was cut and placed in a 1.5 ml microcentrifuge tube using a 3 mm Harris Micro PunchTM (Schleicher & Schuell Bio Science, Keene, NH 03431, USA).
The QIA amp DNA Blood Mini Kit (QIAGEN, DNeasy® Blood & Tissue Kit, Cat. no. 69506, USA) was used to extract DNA from the DBS according to the manufacturer's procedure.
PCR amplifcation and allele detection of msp1 gene
Genomic DNA was amplified using allelic specific primers by nested PCR (Additional files 1): (supplementary Table 1) Table S1. PCR amplification 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 final 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 first 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 final 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-specific 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 final 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 defined as the presence of more than one genotype, whereas a monoclonal infection was defined 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 identified 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), certificate 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 coefficient was calculated. P<0.05 was selected as a threshold for statistically significant differences. The expected heterogeneity (He) was calculated by the formula; \(He=\left(\frac{n}{n-1}\right)\left(1-\sum {p}^{2}\right)\) where “n” stands for the number of the isolates analyzed and “p” represents the frequency of each different allele at a locus [14].