The study was conducted in Ilu Harar Health Center, Chewka district, Buno Bedele Zone, Southwest Ethiopia during September-December 2017. Chewaka district is located in Buno Bedele zone, Oromia regional state, Ethiopia about 570 kilometers southwest of Addis Ababa. It is situated in lowland areas of Dhidhesa valley, which lies below 1500m above sea level. The district has 26 administrative kebeles (villages). As in most other areas, malaria transmission in Chewaka follows rainy seasons, with transmission peaking in the months between September and December and between April and May. The main malaria control strategy in the district includes Long-Lasting Insecticidal Nets (LLINs), indoor residual spraying (IRS) and malaria case management with ACTs .
Study population and blood sample collection
A total of 80 P. falciparum blood spotted samples were collected from study participants aged six months to sixty years, who visited the health center during the evaluation of a therapeutic efficacy study of arthemeter-lumefantrine from September-December, 2017 (submitted for publication at BMC malaria Journal; Manuscript Number: MALJ-D-19-00615). The study participants who were febrile, with an axillary body temperature ≥37.5°C or having history of fever within the previous 24 hours, who fulfilled WHO revised protocol for malaria drug therapeutic efficacy study[38, 39] and signed an informed consent were included in the study. Blood samples were collected on day 0 when patients were enrolled in the study. The blood was spotted onto filter paper (Whatman® 927 mm), air-dried and stored at -20°C, for further parasite genotyping.
Extraction of parasite DNA
Genomic DNA was extracted from whole blood using proteinase K-base method (GE Healthcare Illustra Blood Genomic Prep Mini Spin Kit) according to the manufacturer’s instructions for qPCR species identification and parasite density determination. For nested PCR, the DNA was extracted from stored dried bloodspots collected on enrollment (Day-0) and on any day after day 3 were deemed to have recurrent parasitaemia using Pure LinkTM Genomic DNA mini Kit (Invitrogen, USA) according to the manufacturer’s instructions. DNA was checked for purity and quantity using Nanodrop spectrophotometer (ND 1,000), and stored at -20 °C until used for PCR amplification and detection.
Quantitative PCR (qPCR) screening for Plasmodium falciparum
Primer design genesig standard Kit for Plasmodium spp. (all species) genomes was analyzed for the in vitro quantification of all plasmodium species genomes by targeting the 18S ribosomal RNA (18S) gene according to the protocol of Primer designTM Ltd . Plasmodium falciparum genome was analyzed for the in vitro quantification of P. falciparum genomes by targeting the plasmepsin 4 gene according to the protocol of PrimerdesignTM Ltd . Each reaction was performed in duplicate and the cycle threshold number (Ct) was determined as their mean. A sample was considered positive if the fluorescent signal was detected in at least one replicate; conversely, if no signal was detected within 40 cycles, a reaction was considered negative.
Genotyping of Plasmodium falciparum isolates
Genotyping of P. falciparum isolates was carried out by Nested PCR amplification of the two highly polymorphic regions of msp-1 (block2) and msp-2 (block3) genes as reported previously[42, 43]. Primer sequences(Additional file 3: Table 1) and cycling parameters used for amplification of the three allelic families of msp-1(K1, Mad20 and RO33) and two allelic families of msp-2 (FC27 and 3D7) have been reported elsewhere [44, 45]. Briefly, in the initial amplification, primer pairs corresponding to conserved sequences within the polymorphic regions of each gene were included in separate reactions. The product generated in the initial amplification was used as a template in five separate nested PCR reactions. In the nested reaction, separate primer pairs targeted the respective allelic types of msp-1(K1, Mad20 and RO33) and msp-2 (IC3D7 and FC27), with an amplification mixture containing 250nM of each primer, 2mM of MgCl2 and 125µM of each dNTPs and 0.4 units Taq DNA polymerase (MyTaqTM DNA Polymerase, Bioline). The cycling conditions in the thermocycler (TECHNE, GENIUS), for initial msp-1 and msp-2 PCR were as follows: 5 min at 95 °C, followed by 25 cycles for 1 min at 94 °C, 2 min at 58 °C and 2 min at 72 °C and final extension of 5 min at 72 °C. For msp-1 and msp-2 nested PCR, conditions were as follows: 5 min at 95 °C, followed by 30 cycles for 1 min at 95 °C, 2 min at 61 °C and 2 min at 72 °C and final extension of 5 min at 72 °C. The allelic specific positive control 3D7 and DNA free negative controls were included in each set of reactions . Fragment analysis of msp-1 and msp-2 amplified products were then performed through electrophoresis on 2% and 3% ethidium bromide-stained agarose gel, respectively, and the fragments were visualized under UV light. Gel photographs were re-scored by visual comparison of DNA fragments and for individual samples, alleles were identified according to band size (Additional file 2: Figure S1; Additional file 3: Figure S2). The size of the PCR products was estimated using 100bp DNA ladder marker. We assessed the frequency of the occurrence of each allele in the population. We categorized clones into molecular weight groups differing by 20bp for clear discrimination from other clones and elimination of errors that would result from estimating the molecular weight on agarose-gels.
Samples from participants with multi-clonal infection produced more than one amplified fragment after either the three msp-1 or the two msp-2 allelic family PCR reactions . Samples from participants with clonal infection produced a single product after the three msp-1 PCR reactions as well as a single product after the two msp-2 reactions. The msp-1 and msp-2 allele frequencies were expressed as the proportion of samples containing an allelic family compared to the total number of samples that gene was detected in isolates. The detection of one msp-1 and msp-2 allele was considered as one parasite genotype. The multiplicity of infection (MOI) was defined as the minimum number of P. falciparum genotypes per infected subject and estimated by dividing the number of amplified PCR fragments reflecting the parasite genotypes by the number of positive samples in the same marker . The size of polymorphism in each allelic family was analyzed; assuming that one band represented one amplified PCR fragment derived from a single copy of P. falciparum msp-1or msp-2 genes. Alleles in each family were considered the same if fragment sizes were within 20bp interval . Spearman’s rank correlation coefficients were calculated to assess association between multiplicity of infection (MOI) and geometric mean parasite density and age. The heterozygosity index (He), which represents the probability of being infected by two parasites with different alleles at a given locus, was calculated by using the Genetic Analysis in Excel toolkit (GenAIEx) . Briefly, the allelic diversity (He) for each antigenic markers was calculated based on the allele frequencies, using the formula: He= [n/ (n-1)] [(1-Σpi2)], where n is the number of isolates sampled and pi is the allele frequency at a given locus. Allelic diversity has a potential range from 0 (no allele diversity) to 1 (all sampled alleles are different) . All statistical analyses were performed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant if p-value < 0.05.