The main objective of this study was to evaluate the prevalence in Benin of P. falciparum resistance markers to CQ and SP, two conventional anti-malarials that have been used for a long time. This study was justified by the need of surveillance of P. falciparum resistance to anti-malarials drugs in Benin through well-characterized molecular markers of resistance to contribute to the monitoring in the subregion. This monitoring is intended on the one hand to watch for a possible return of sensitivity of P. falciparum to CQ and secondly, to describe the current patterns of molecular markers associated with parasite resistance to SP, the latter still used as a preventive treatment of malaria in pregnant women. This work was carried out on parasites obtained from two distinct clinical groups of children, to investigate a possible relationship between molecular markers and clinical status. Mutation at codon 164 of pfdhfr gene has not been studied because several studies have noted the absence or rarity of this mutation in African isolates
High rates of single, double, triple or quadruple mutation observed in this study reflect the current level of sensitivity of P. falciparum to CQ and SP in Benin, which is still expected to be low although the two drugs were officially withdrawn from curative treatment of uncomplicated malaria in 2004. These high rates of mutant parasites had been previously found by other authors
[23, 25–28]. The fact that the frequency of mutations at codon site 437 of pfdhps is lower than those observed on the pfdhfr gene confirms that the mutations associated with parasite resistance to SP appeared earlier on the pfdhfr than those affecting the pfdhps[29, 30].
Similar to other reports from the sub-region
[31, 32], the parasites harbouring the mutation at codon 540E were not found in Benin. Regarding the pfcrt gene, analysis of the T76 mutation (93.9%) in isolates from Benin showed a high prevalence of parasites carrying this mutation. Unlike Malawi, where there has been a reversal of the prevalence of mutant T76 a few years after the withdrawal of CQ
[10, 11], it is clear that this has not been the case in Benin. Self-medication with fake medicines especially with regard to CQ (despite its formal withdrawal from the treatment policy of malaria) could be a leading cause for maintaining this high prevalence of T76 mutant parasites as a result of continued and frequent use of CQ
 involving either insufficient doses or too short a duration of administration. It is a common practice in the south of the country characterized by large markets (with neighbouring Nigeria) where illegitimate distribution of fake drugs is common
[2, 34]. This could also be marginally explained by a possible cross-resistance shared between CQ and AQ
[35, 36], since AQ is present in the ASAQ combination currently used for malaria management in Benin. WHO in vivo drug efficacy studies conducted in 2008 with this combination in two localities of the country noted an adequate clinical and parasitological response (ACPR) of 100% and 76.6% respectively without PCR correction. The second locality (Dassa Zoume) with a decrease of ACPR (23.44% of late parasitological failure) had posted 38.8% of late parasitological failure in 2002 during in vivo drug efficacy studies with CQ (unpublished data from Ministry of Health, Benin).
Thus, the re-emergence of sensitive parasite strains after the withdrawal of CQ depends not only on the time elapsed since the withdrawal of the treatment, but also on the speed of replacement and implementation of the new drugs, to allow the cessation of use and consumption of drugs replaced. It is important that the health authorities of the country ensure the effective withdrawal of CQ by emphasizing the education about the risks of self-medication and implementing control methods against the entry of fake medicines. The results obtained with the gene pfcrt are consistent with those reported in China by Zang et al., but is in contrast to those reported by Kamugisha et al. in Tanzania
 and Raman et al. in Mozambique
Regarding SP, it should be noted that the use of other sulpha drugs, such as trimethoprim-sulphametoxazole (co-trimoxazole) often prescribed for bacterial infections in children but also in prevention and treatment of opportunistic infections in persons living with HIV, contribute in maintaining drug pressure on malaria parasites. The use of SP in intermittent preventive treatment in pregnant women (IPTp) since its withdrawal from the treatment of uncomplicated malaria may also explain the maintain of the selective pressure on SP targets as shown in the study conducted by Bertin et al. in Benin and Pearce et al. in Tanzania
[13, 40]. Similarly to what has been reported by other authors
[17, 27], this study shows that regardless of the gene, mutations are not associated with the clinical status of children. On the other hand, to determine whether immunity contributed to the ability to clear infections by parasites carrying resistance, the proportion of infections by parasites carrying different mutations compared between children younger than 10 years and older children suggests that age does not influence the distribution and carriage of resistant parasites whatever the clinical status and type of mutation.