This study highlights the real burden of P. vivax infections by updating prevalence data in eight sites throughout Madagascar. Despite the likely underestimation of the prevalence of P. vivax due to the use of the RDT for malaria screening , this study confirms, however, that P. vivax is the second prevalent species in malaria infections after P. falciparum. The highest prevalence of Plasmodium vivax infections were found in the western side of Madagascar, certainly because of the predominance of ethnic groups of Indo-Asian and Middle Eastern origin, who carry the Duffy antigen .
SP resistance and related single nucleotide polymorphisms (SNPs) in the pvdhfr and pvdhps genes were analysed in P. vivax samples from seven sites representative of the four major epidemiological strata for malaria. The two genes were screened by sequencing, to identify possible new mutations. The P. vivax pvdhfr gene is known to be highly diverse, supporting the use of such an approach . A very high proportion of mutant-type isolates (72.2%) and diverse alleles were identified in these isolates, with 15 different mutant-type alleles observed if both pvdhfr and pvdhps gene mutations were taken into account. This is the first time that mutations at positions implicated in SP resistance have been described in P. vivax isolates from Madagascar.
Only two substitutions were identified in pvdhps, whereas six non-synonymous mutations, including four that have already been described, were found in pvdhfr. Mutations in these two genes do not play identical roles in the emergence of SP-resistance. Similar observations have been made for P. falciparum [28, 29]. Mutations seem occurring first in pfdhfr gene, then after in pfdhps gene when most of the parasites in the population have double- or triple-mutant alleles of the dhfr gene .
Mutations in pvdhfr, including 58R and 117N, have been implicated in pyrimethamine resistance. The 58R allele was found in 58% of all P. vivax isolates, in combination with 117N, which was found alone or in combination with other mutations in 63.5% of all isolates. Studies in vitro have shown that the mutation of a single base, leading to the replacement of a serine by an asparagine residue at codon 117, increases the IC50 value of pyrimethamine by more than 80 times. The combination of this mutation with the replacement of a serine by an arginine residue at codon 58 generates an enzyme more than 400 times more resistant to pyrimethamine than the wild-type enzyme [30, 31]. The 57L/58R/117N, triple mutant previously observed in Thailand  and associated with low levels of parasite clearance was not found. No parasites of the 57L/58R/61M/117T quadruple mutant type, associated with a high risk of SP treatment failure, were also not found.
Two new mutations were found at codons 21 and 130 in the pvdhfr gene. The mutation at codon 130 accounted for 31.4% of the isolates. This mutation, present mostly at Miandrivazo (67% of isolates), was strongly associated with the 58R/117N double mutant. The P33L substitution accounted for only 6% of the isolates. This mutation was previously found to be associated with isolates of Comorian or Malagasy origin [14, 25]. One of the unique features of the pvdhfr gene of P. vivax is the presence of a tandem repeat between amino-acid residues 70 and 110. Size polymorphism has been reported in this region [22, 32], but no variation was observed in this study.
It was impossible to assess the impact of the new N130K mutation based on clinical data, because only the 58R/117N double mutant was observed in isolates from the patients enrolled in the clinical trial of SP efficacy, including the isolate with the A383G mutation in pvdhps. The use of yeast constructs might facilitate interpretation of the role of this mutation in resistance .
Because of the absence of a genotyping consensus protocol for P. vivax to differentiate recrudescence from reinfection, the use of a combination of different genes such as pvama1, pvmsp1, pvmsp3 or microsatellite markers in paired analysis seems to be the safest available method. In this study, pvcsp and pvmsp3 genes sequencing and six different microsatellite markers were used on samples from day 0, day 1 and day of reoccurrence. The use of microsatellite markers seems to be useful as more polyclonal infections could be detected. Obvioulsy, the main limitation of this protocol was the well-recognized impossibility to prove that a P. vivax reoccurrence was a recrudescence, a relapse or a reinfection.
No significant association between pvdhfr/pvdhps polymorphisms and SP-treatment outcome was found, but all recrudescent patients were pvdhfr double-mutant carrier. This result strongly suggests that infection due to the pvdhfr 58R/117N double mutant is necessary but not sufficient for SP treatment failure to occur. The treatment outcome is likely to be favourable if the parasite has a wild-type genotype, but in parasites with mutations, outcome depends on the alleles present at the pvdhfr and pvdhps loci and the individual response of the patient . Currently, it is well known that beside parasite factors, host factors such as nutritional status, immune response and rates of drug metabolism are involved in determining treatment outcome.
Surprisingly, almost 72.3% of the tested P. vivax isolates had mutations in the pvdhfr and/or pvdhps genes, despite SP never having been recommended as a first-line treatment for malaria, but only as a second-line treatment from 1998 to 2005. ACT is now recommended for the treatment of uncomplicated malaria regardless of the causal Plasmodium species. Nevertheless, self-treatment remains frequent in Madagascar since unpublished study has shown that three quarters of all febrile patients attending government health facilities have already used chloroquine in 67.6% of cases, cotrimoxazole (sulphamethoxazol/trimethoprim) in 23.4% and SP in 9.4% of cases. Cotrimoxazole is the drug most widely used to treat diarrhoea as well as respiratory infections . Asymptomatic P. vivax infections and treatment with trimethoprim are probably common, resulting in the exposure of parasites to this drug. Mutations in the pvdhfr and pvdhps genes may reflect overall antifolate drug pressure in Madagascar.
Previous studies have shown that dhfr mutant P. falciparum isolates were extremely rare , with only one case of infection with the 108N mutant reported in the south of Madagascar. Based the observations for P. vivax isolates, data for P. falciparum genotypes should be updated.