Use of saliva for monitoring Plasmodium falciparum chemoresistance to pyrimethamine in three sites in Southern Cte Divoire

Current malaria diagnostic methods require blood sampling, which can be a barrier due to needle-related discomfort or cultural taboos surrounding blood. Saliva, being minimally invasive to collect, offers a promising alternative. This study aims to utilize saliva to monitor Plasmodium falciparum resistance to pyrimethamine at three sites in southern Côte d'Ivoire. Blood and saliva samples were collected from 94 patients over 2 years old with microscopically confirmed uncomplicated P. falciparum malaria across three localities. The genomic DNA of P. falciparum was extracted, amplified using nested PCR with primers specific to the pfdhfr gene, and sequenced using the Sanger method. The sequencing results were then analyzed to determine the prevalence of pfdhfr mutations (N51I, C59R, S108N) associated with pyrimethamine resistance in P. falciparum. Data analysis was performed using R statistical software, version 3.2.2. A total of 153 DNA fragments of the pfdhfr gene were sequenced, comprising 86 blood DNA fragments and 67 salivary DNA fragments. Successful sequencing rates for blood DNA fragments were 75.58% (N51I), 76.74% (C59R), and 94.18% (S108N), compared to 85.07% (N51I), 86.56% (C59R), and 94.02% (S108N) for salivary DNA fragments. Sequence analysis revealed mutation prevalences in the pfdhfr gene of 61.53% (N51I), 54.54% (C59R), and 74.07% (S108N) in blood, and 49.12% (N51I), 63.79% (C59R), and 79.36% (S108N) in saliva. Notably, no significant difference was observed between the mutation prevalences in blood and saliva (p = 0.44). Molecular analysis revealed that the sensitive NCS haplotype (51N59C108S) was detected in 17 out of 153 isolates, with a prevalence of 13.96% (12/86) in blood and 7.46% (5/67) in saliva. In contrast, the IRN triple mutant haplotype (51I59R108N) was observed in 48 out of 153 isolates, with a prevalence of 31.4% (27/86) in blood and 31.34% (21/67) in saliva. Notably, the prevalences of the IRN triple mutant haplotype did not differ significantly between blood and saliva (p = 0.685). This study demonstrated that the prevalence of genotypes conferring resistance to pyrimethamine reached comparable levels in both blood and saliva isolates. Over a decade after the adoption of sulfadoxine-pyrimethamine as intermittent preventive treatment for pregnant women, the prevalence of the Asn-108 allele and the triple mutant IRN haplotype remained relatively high in Anonkoua-kouté, Port-Bouët, and Ayamé, Côte d'Ivoire.   Key words: Pfdhfr, saliva, Côte d'Ivoire, sulfadoxine-pyrimethamine, resistance, antimalarial drugs, Plasmodium falciparum.