Demographic characteristics
A total of 972 outpatients were screened for malaria, of which 197 (20.3%) were RDT positive. Ninety-eight (49.7%) of the RDT positive cases were confirmed by microscopy to be P. falciparum positive (Fig. 1). Eighty-four (85.7%) patients with RDT and microscopy positive results with at least 2000 parasites/uL were enrolled and followed up for 28 days, of which seventy-one (84.5%) of the enrolled participants completed follow-up. The enrolled patients were categorized into three age groups; <5 years (8 patients; 9.5%), 5a-15 years (57 patients; 67.9%), and >15 years (19 patients; 22.6%) (Table 1). The positive samples recorded by microscopy on days 0, 1, 3, 7, 14, 21 and 28 were 84/84 (100%), 36/80 (45%), 0/78 (0%), 2/76 (3%), 0/75 (0%), 0/72 (0%), and 2/71 (3%) respectively, given that thirteen (16%) participants were lost at different times to follow-up (Fig. 1). Geometric mean parasitaemia at baseline was 42,727 parasites/μL (95% CI 14,522 – 26,599) while the mean axillary temperature was 37.4°C (95% CI 37.1 – 37.7). There was an inverse relationship between age and parasite density (correlation coefficient = -0.2072, P = 0.0586) (Fig. 2; Additional File 1: Fig. S1). The same relationship was observed for age vs axillary temperature (correlation coefficient = -0.1626, P = 0.1394) (Additional File 1: Fig. S2).
18S rRNA diagnostic PCR and treatment outcome
Eighty-four day 0 samples from the enrolled patients were screened by 18S rRNA PCR, of which 82 (97.6%) were confirmed P. falciparum-positive (Additional File 1: Plate S1). On follow-up days, 31/78 (39.8%), 23/76 (30.3%), 5/74 (6.8%), 4/73 (5.5%), 1/70 (1.4%) and 2/69 (2.9%) were 18S rRNA PCR-positive on days 1, 3, 7, 14, 21 and 28 respectively (Table 2). According to the PCR-uncorrected data, 4/71 (5.6%) patients were classified as LPF, while 67/71 (94.4%) patients achieved ACPR in the per-protocol analysis (Table 3). However, after PCR correction, no suspected LPF case was detected and ACPR was observed in 67/67 (100%) individuals (Table 3; Additional File 2: Sheet S2).
MSP1 and MSP2 allelic diversity
MSP1 genotyping was achieved for 61 isolates while 45 isolates were typed for MSP2 locus. For MSP1 isolates, three K1 (160-500 base pairs), five MAD20 (220-500 bp) and five RO33 (160-600 bp) alleles were identified (Additional File 1: Plate S2; Additional File 2: Sheet S7). The predominant MSP1 allelic family was K1 55/61 (90.2%), followed by RO33 38/61 (62.3%) and MAD20 31/61 (50.8%). Specifically, 19/61 (31.1%) of MSP1 positive samples had only K1 family, 9/61 (14.8%) had MAD20, and 7/61 (11.5%) had RO33 allelic family. Monoclonal infections were 35/61 (57.3%), while the remaining 26 (42.6%) were polyclonal, with K1/MAD20, K1/RO33, and MAD20/RO33 representing 6/61 (9.8%), 11/61 (18.0%), and 1/61 (1.6%), respectively (Fig. 3). Furthermore, 8/61 (13.1%) samples possessed three MSP1 allelic types (i.e., trimorphic). Eight MSP2 alleles were detected, including three FC27 and five 3D7 alleles, with sizes ranging between 400 to 800 bp for FC27 allelic family and 300 to 800 bp for 3D7 (Additional File 1: Plate S3). The frequency of FC27 and 3D7 were 29/45 (64.4%) and 26/45 (57.8%), respectively (Additional File 1: Table S5); 20/45 (44.4%) had only FC27, 17/45 (37.8%) had only 3D7 while 8/45 (17.8%) possessed both MSP2 allelic families (Fig. 4). The estimated MOIs were 2.0 and 1.2 for MSP1 and MSP2 loci respectively (Additional File 1: Table S5). The overall mean MOI was 1.7. There was no statistically significant correlation between age and MOI estimated for MSP1 and MSP2 (Spearman rank coefficient = 0.5000; P > 0.9999) loci (Table 4). However, the highest MOI was observed in the 5 – 15 years group (2.1 for MSP1 and 1.2 for MSP2). Also, no significant correlation was observed between MOI and the parasitemia for MSP1 (Spearman rank coefficient = 0.5000; P > 0.9999) and MSP2 (Spearman rank coefficient = 1.0000; P = 0.3333) markers (Table 5).
Prevalence of MDR1 mutations
The prevalence of N86Y and D1246Y mutations was determined for 64 PCR positive samples. For MDR1-N86Y locus, we found a prevalence of 60/64 (93.7%) for wild-type MDR1-N86 (Table 6). No mutant or mixed MDR1-86Y allele was found. With respect to MDR1-D1246Y locus, the prevalence of mixed mutant and wildtype genotype was 4/64 (6.2%), 58/64 (87.5%) wildtype D1246, and 1/64 (1.6%) had 1246Y mutant allele. Overall, there was a high proportion of circulating parasites with the wild type alleles for MDR1 genes.
UBP1 polymorphisms
Sixty-one P. falciparum isolates with positive PCR amplification of the 304-bp region were Sanger sequenced (Additional File 1: Plate S4). Among these, one isolate (1.6%) was found to harbor one non-synonymous mutation (Tables 7 and 8). In total, seven indels were identified, with 11.5% (7/61) causing a frameshift in the sequence reading frame. Multiple nucleotide sequence alignments revealed that three isolates (4.9%) had AAATATGAT deletions (encoding KYD) at amino acid residues 1532 to 1534. Furthermore, two isolates had insertions of AAATATGAT (encoding KYD) at amino acid residues 1535 to 1537, one insertion at amino acid residues 1528 to 1530 and one at 1532-1534 (Table 7). A single nucleotide substitution (1/61; 1.6%) was also observed in 1528 locus (Table 7).