This cross-sectional study was conducted at the Regional Center for Mutuality and Social Welfare in School Settings (CREMOSS) of Daloa, located in the Haut-Sassandra region in west-central Côte d’Ivoire, from April to June 2025. The study site is a referral and primary healthcare facility receiving patients from various districts of the city and surrounding localities, thereby providing access to a heterogeneous population.

The study population comprised patients of all age groups who were permanent residents of the Haut-Sassandra region and had been prescribed laboratory testing for malaria diagnosis. All samples meeting the inclusion criteria and available during the study period were consecutively enrolled. The sample size was determined by the number of eligible participants attending the hospital during the recruitment period and by the availability of biological samples for molecular analyses.

Participants were included in the study if they were permanent residents of the Haut-Sassandra region, presented with suspected clinical malaria requiring a medical prescription for parasitological examination, and provided informed consent. For minors, assent along with parental or guardian consent was required.

Patients were excluded if they refused to participate, had received antimalarial treatment within the two weeks prior to recruitment, or presented incomplete clinical or biological data.

Following the interview and clinical assessment, 5 mL of venous blood was collected from each participant into EDTA-containing tubes by trained laboratory personnel. Samples were properly labeled and immediately transported to the laboratory under conditions ensuring an appropriate cold chain and full traceability of specimens. Thick blood smears were prepared on clean, grease-free slides, air-dried, and then stained with 10% Giemsa solution. Microscopic examination was performed by an experienced microscopist following World Health Organization (WHO) standardized procedures, using a light microscope with oil immersion (×100 objective). Malaria parasitaemia was quantified by counting asexual parasite forms per 200 leukocytes and expressed as parasites/µL, using a standard leukocyte count of 8,000/µL.

Internal quality control was ensured through systematic re-examination of all positive slides and a random subset of negative slides (10%) to verify consistency and reliability of the results. In cases of uncertainty in slide interpretation, a repeat examination was performed for confirmation. In addition, 2 mL aliquots of whole blood were collected from each participant, independently of microscopy results, and stored in uniquely labeled cryotubes at −20˚C for subsequent molecular analyses.

Participants were informed about the objectives and methodology of the study in French or in a local language. Written informed consent was obtained from all adult participants, while parental consent and child assent were obtained for minors. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. Participant confidentiality was strictly maintained throughout the study.

Genomic DNA was extracted from 200 µL of venous blood using the commercial QIAamp DNA Blood Mini Kit (QIAGEN, Germany), according to the manufacturer’s instructions.

Molecular detection of P. falciparum was performed by amplification of the 18S ribosomal gene (small subunit ribosomal RNA, SSU rRNA) using nested PCR. Each PCR run systematically included a positive control consisting of reference P. falciparum DNA confirmed to carry the target alleles, as well as a negative control (sterile distilled water) to detect any potential contamination. The first amplification was carried out using a pair of primers targeting conserved regions of the 18S gene of Plasmodium species (Table 1). The resulting products were then used as templates for a second amplification using Plasmodium falciparum-specific primers (Table 1).

PCR reactions were performed in a final volume of 25 µL containing 4 µL of DNA template (1 ng/µL), 1 µL of each primer (10 pmol/µL; Eurogentec, France), 12.5 µL of GoTaq Green Master Mix (Promega, USA), and nuclease-free sterile water to adjust the final volume.

The thermal cycling program for the first PCR consisted of an initial denaturation at 95˚C for 5 min, followed by 45 cycles of denaturation at 94˚C for 1 min, annealing at 57˚C for 2 min, and extension at 72˚C for 2 min, with a final extension at 72˚C for 5 min. The second amplification was performed under the same reaction conditions using 4 µL of the first PCR product as template. Cycling conditions included an initial denaturation at 95˚C for 5 min, followed by 45 cycles consisting of denaturation at 94˚C for 1 min, annealing at 55˚C for 2 min, and extension at 72˚C for 2 min. A final extension was carried out at 72˚C for 5 min. All amplifications were performed using a conventional thermocycler (Bio-Rad, USA).

Second-round PCR products were analyzed by electrophoresis on a 2% agarose gel containing GelRed and visualized under ultraviolet light. Amplification product sizes were estimated by comparison with a 100 bp molecular weight marker. A band of approximately 1200 bp was interpreted as positive for the Plasmodium genus, whereas a 205 bp fragment was specific for Plasmodium falciparum.

Table 1. List of primers used for the diagnosis of Plasmodium falciparum [11].

Note: bp: base pair.

Confirmed Plasmodium falciparum-positive samples were used for the amplification of Pfmdr1 gene polymorphisms at codons 86 and 184. The selection of codons 86 and 184 of the pfmdr1 gene is justified by their central role in modulating susceptibility to major antimalarial drugs used in Africa, including chloroquine, amodiaquine, and artemisinin-based combination therapies. These two loci are highly informative markers in molecular epidemiology, as they exhibit substantial allelic variability and are frequently involved in drug pressure-driven selection processes. In contrast, the D1246Y codon, although biologically relevant, is less prevalent in several West African populations, which limits its discriminative power in studies with moderate sample sizes [12]. Amplification was performed using nested PCR with two sets of specific primers, A1/A3 for the primary PCR and A2/A4 for the secondary PCR (Table 2), according to the method described by [13]. Each PCR run systematically included a positive control consisting of reference P. falciparum DNA confirmed to carry the target alleles, as well as a negative control (sterile distilled water) to detect any potential contamination. PCR reactions were performed under conditions identical to those previously described. The thermal cycling program included an initial denaturation at 94˚C for 2 min, followed by 40 cycles of denaturation at 94˚C for 1 min, annealing at 45˚C for 1 min, and extension at 72˚C for 1 min, with a final extension step at 72˚C for 5 min.

Table 2. Primers and amplification conditions for Pfmdr1 codons 86 and 184.

Note: bp: base pair.

Genotyping of the Pfmdr1 N86Y and Y184F polymorphisms was performed using restriction fragment length polymorphism PCR (PCR-RFLP). The secondary PCR products were digested with the restriction enzymes ApoI for codon 86 and DraI for codon 184, in a final volume of 15 µL at 37˚C for 1 h (Table 3), according to the method described by [14]. The restriction fragments were then separated by electrophoresis on a 2% agarose gel at 60 V for 1 h. The resulting migration profiles allowed discrimination of wild-type, mutant, and mixed genotypes based on fragment sizes. For the N86Y polymorphism, isolates carrying the wild-type N86 allele showed a single 560 bp band, whereas mutant 86Y isolates exhibited a digested fragment characterized by a 333 bp band. Mixed infections were defined by the simultaneous presence of bands corresponding to both alleles (560 bp and 333 bp). Regarding the Y184F polymorphism, wild-type isolates showed an undigested single band, whereas mutant isolates displayed three fragments of distinct sizes after DraI digestion, consistent with the expected profile. Mixed infections were defined by the simultaneous presence of the undigested wild-type profile and the digested mutant profile after DraI treatment. However, mixed infections were excluded from the final analysis due to the limited resolution of PCR-RFLP for reliably discriminating co-existing allelic profiles, in order to ensure the robustness of allele frequency estimates.

Table 3. Digestion conditions of ApoI and DraI enzymes.

Data were entered, verified, and cleaned according to standard data management procedures prior to statistical analysis. Participant characteristics were described using descriptive statistics. pfmdr1 gene genotypes were determined based on the presence or absence of wild-type and mutant alleles at the different loci investigated. Mutation frequencies were estimated by grouping isolates carrying mutant alleles.

Associations between categorical variables and malaria infection were assessed using Pearson’s Chi-square test or Fisher’s exact test when expected counts were less than 5. Differences were considered statistically significant at a p-value < 0.05, with a 95% confidence interval.

Non-informative genotypes, corresponding to samples showing weak, ambiguous, or non-interpretable banding patterns after PCR-RFLP electrophoresis, were excluded from the final analysis. This exclusion was performed to minimize interpretation bias associated with poor resolution of restriction fragments and to ensure the reliability and robustness of allele frequency estimates and statistical analyses.

A total of 179 participants were included in this study, comprising 111 (62%) females and 68 (38%) males, corresponding to a sex ratio (M/F) of 0.61 (Table 4). Molecular analyses revealed an overall prevalence of Plasmodium falciparum infection of 50.27% (Table 4). Statistical analyses showed a significant association between age and malaria infection (p = 0.036; Table 4), with a notable difference between children aged 6 - 15 years and adults (>15 years). Moreover, children aged 6 - 15 years had a 2.08-fold higher risk of malaria infection compared to adults.

Table 4. General characteristics.

Note: n: Study population, F: Female, M: Male, IC 95%: 95% confidence interval, OR: Odd ratio, *: significant result.

Genotyping of codon 86 of the pfmdr1 gene was successfully performed on all 90 isolates, whereas that of codon 184 was obtained for 80 isolates due to amplification failure at this codon (Table 5).

Analysis of Pfmdr1 gene polymorphisms revealed a predominance of mutant alleles in the studied isolates. At codon 86, the mutant allele 86Y was detected in 72.2% of samples compared to 27.8% for the wild-type allele N86. Similarly, at codon 184, the mutant allele 184F accounted for 85.0% of isolates, whereas the wild-type allele Y184 was found in only 15.0% of samples.

Table 5. Frequency of genotypes in the pfmdr1 gene.

Note: IC 95%: 95% confidence interval.

According to sex, a higher prevalence of the mutant allele 86Y was observed in males (76.47%) compared to females (69.64%) (Table 6). However, this difference was not statistically significant (OR = 1.42; 95% CI: 0.53 - 3.76; p = 0.484). A similar trend was observed at codon 184, where the frequency of the mutant allele 184F was slightly higher in males (87.5%) than in females (83.33%), with no statistically significant association (OR = 1.40; 95% CI: 0.38 - 5.10; p = 0.61) (Table 6).

Age-based analysis showed that individuals aged 6 - 15 years had the highest frequency of the mutant allele 86Y (82.5%). Compared with subjects older than 15 years, this age group had a 2.46-fold higher risk of carrying the mutant allele, although this association did not reach statistical significance (OR = 2.46; 95% CI: 0.84 - 7.20; p = 0.10). In contrast, children aged 0 - 5 years showed a lower frequency of the 86Y mutant (60%), with no statistically significant difference (OR = 0.78; 95% CI: 0.22 - 2.72; p = 0.70) (Table 6).

For codon 184, the frequency of the mutant allele 184F remained high across all age groups, with prevalences of 73.33% in children aged 0 - 5 years, 88.23% in those aged 6 - 15 years, and 87.09% in individuals older than 15 years. No statistically significant association was observed between age groups and carriage of the 184F mutant allele (p > 0.05) (Table 6).

Overall, mutant alleles of the Pfmdr1 gene were highly predominant in the study population, with no significant association with sex or age groups. Nevertheless, the high frequency of the 86Y and 184F mutants may reflect persistent antimalarial drug selection pressure in the study area.

Table 6. Analysis of Pfmdr1 gene polymorphisms (codons 86 and 184) according to sex and age groups.

Note: OR: Odd ratio, M: Male, F: Female, IC 95%: 95% confidence interval.

Haplotype analysis of the pfmdr1 gene revealed a predominance of the double mutant haplotype 86Y-184F, accounting for 65.00% of successfully genotyped isolates (Table 7). The single mutant haplotype N86-184F represented 20.00%, while the double wild-type haplotype N86-Y184 was observed in only 13.75% of isolates (Table 7). The low prevalence of the 86Y-Y184 haplotype suggests a preferential co-selection of the 86Y and 184F mutant alleles within the local parasite population.

Table 7. Haplotype analysis of pfmdr1 gene mutations.

Note: WT: wild-type, M: Mutant.