In Senegal malaria remains a formidable public
In Senegal, malaria remains a formidable public health issue, causing significant morbidity and mortality in infants and pregnant women (WHO Malaria Report, 2012). In the absence of an effective vaccine, the National Malaria Control Program has followed WHO recommendations for IPTp-SP since 2003. The rapid spread of SP-resistant parasites highlights the need for regular monitoring of ex vivo parasite sensitivity to pyrimethamine and dhfr/dhps mutations in countries like Senegal, where SP has been widely used for several years.
Materials and methods
Discussion In 2003, Senegal adopted intermittent preventive treatment for pregnant women (IPTp) using sulfadoxine–pyrimethamine (SP). At the same time, between 2003 and 2004, Senegal switched to sulfadoxine–pyrimethamine with amodiaquine as the first-line therapy for uncomplicated malaria in response to increasing chloroquine resistance (WHO Roll Back Malaria Focus on Senegal, 2010). In 2005, Senegal adopted artemisinin combination therapies (ACTs) as first line treatment for uncomplicated malaria. The results reported here were obtained from samples collected from the general population in an urban site with expanded SP use, and this is one of the few reports that includes both dhfr/dhps polymorphisms and ex vivo drug phenotype data. Previous studies carried out in Senegal and other West African countries have focused on rural sites and studied P. falciparum polymorphisms without assessment of corresponding ex vivo phenotypes. Our results show an increase in the prevalence of parasites bearing individual dhfr mutations at codons 51, 59, and 108 in an interval of eight years. Furthermore, the number of parasites with all three dhfr mutations increased from 40% in 2003 (Ndiaye et al., 2005), to 93% in 2011. Emergence of the dhfr 51I/59R/108N triple mutant has been observed in countries using sulfadoxine–pyrimethamine alone or in combination, as first line treatment for uncomplicated malaria as reported in Africa and elsewhere (Bwijo et al., 2003; Griffin et al., 2010; Malisa et al., 2010; Raman et al., 2010; Yusuf et al., 2010, Zakeri et al., 2010, Mula et al., 2011, Mombo-Ngoma et al., 2011, Naidoo and Ropper, 2011). A similar increase in the dhfr N51I/C59R/S108N triple pim kinase inhibitor has been observed after IPT in children in southern Senegal (Faye et al., 2011), as well as in rural regions in Mali (Dicko et al., 2010) and southern Mozambique (Enosse et al., 2008), with dhfr mutations being an important predictive risk factor of in vivo resistance (Boumbou-Moukoko et al., 2009, Picot et al., 2009). Dhps mutations individually fluctuated (no significant change) between 2003 and 2011 in this study, but when considered in combination with dhfr mutations, the number of parasites with an additional mutation at dhps 437 (dhfr N51I/C59R/S108N and dhps A437G quadruple mutation) increased from 2003 to 2008 and then steadily decreased until 2011. Interestingly, we found that mutations at dhps codons 436 and 437 were not always inherited together, despite residing very close to each other on the chromosome (Bwijo et al., 2003; Bouyou-Akotet et al., 2010). The quintuple mutant dhfr 51I/59R/108N and dhps 437G/ 540E has not been previously observed in Senegal (Ndiaye et al., 2005, Ndiaye et al., 2006, Henry et al., 2006; Faye et al., 2011), or in Mali (Dicko et al., 2010). The dhfr I164L mutation was also not found in this study. The combination of dhfr C59R and dhps K540E mutations, which predict clinical failure of sulfadoxine–pyrimethamine (Basco et al., 2000, Kublin et al., 2002, Talisuna et al., 2004, McCollum et al., 2012), were also not found in our study. We found a correlation between the dhfr S108N single mutation and pyrimethamine resistance, and a correlation between the dhfr N51I/C59R/S108N triple mutation, as well as the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation, and pyrimethamine resistance. Overall, we found a significant difference in the geometric mean IC50 values for pyrimethamine (p=0.0009) between parasites possessing wild-type and resistant alleles in dhfr, as has been reported by others (Andriantsoanirina et al., 2011). We confirmed the existence of an association between the dhfr genotype and chemosensitivity to pyrimethamine in P. falciparum isolates from Thies, as the increase in the number of mutations was associated with an increase in ex vivo resistance to pyrimethamine, similar to what has been observed in Gabon (Aubouy et al., 2003), Central African Republic (Menard et al., 2006), and Cote D’Ivoire (Djaman et al., 2007). However, some parasites harbored the N51I, C59R, and S108N mutations in dhfr but were still susceptible to pyrimethamine as reported in isolates from Brazil (Petersen et al., 1991), and Gabon (Aubouy et al., 2003) for the dhfr S108N mutation and Papua New Guinea (Reeder et al., 1996) for dhfr S108N and dhfr C59R. Further sequencing of these parasites for possible compensatory mutations may explain this finding. The ex vivo assay data does not permit strong conclusions because we obtained ex vivo pyrimethamine data from only 1year; however, the high rates of pyrimethamine ex vivo resistance in this study are correlated with high rates of the dhfr N51I/C59R/S108N triple mutation.