Reduced Impact of Pyrimethamine Drug Pressure on Plasmodium malariae Dihydrofolate Reductase Gene

Abstract

Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax; observed in three Cambodian samples). Currently, in many parts of the globe, the emergence and spread of malaria parasites resistant to various antimalarial drugs recommended by the international organizations remain major factors threatening control efforts (41). Among the five Plasmodium species that affect humans (40), resistant Plasmodium falciparum parasites were first selected by chloroquine (CQ), a highly effective, fast-acting, and inexpensive 4-aminoquinoline widely used for several decades, in Southeast Asia or South America (29,44) in the 1960s. CQ-resistant parasites of the other Plasmodium species emerged much later: in 1989 for Plasmodium vivax (Papua New Guinea) (32) and in 2002 for Plasmodium malariae (Indonesia) (26). Following the introduction of the sulfadoxinepyrimethamine combination (SP) to replace CQ as the first-line treatment for uncomplicated P. falciparum malaria, the same scenario was observed. SP-resistant P. falciparum parasites were first detected in the 1980s (9, 19). Molecular investigations, based on laboratory and field isolates, demonstrated later that the resistance of P. falciparum to pyrimethamine was mediated by specific point mutations in the dihydrofolate reductase gene (dhfr) (30, 31). Currently, it is assumed that pyrimethamine resistance is conferred by the stepwise selection of a series of nonsynonymous point mutations (codons 50, 51, 59, and 164) from the S108N single mutant allele (24). Parasites with the triple mutant allele (N51I C59R S108N) have markedly reduced in vitro susceptibility to pyrimethamine, and the presence of this allele in a P. falciparum-infected patient increases the risk of SP therapeutic failure (15). The ...

Genetic Variability of Plasmodium malariae dihydropteroate synthase (dhps) in Four Asian Countries

“…Although patients infected with P. malariae are usually not treated with antifolate drugs, exposure is still likely in endemic areas where sulfadoxine - pyrimethamine is used for treatment of falciparum malaria, because of the high frequency of mixed infections. The previously reported S114N mutation within the dhfr gene of P. malariae supports this [5] , [6] . The P. malariae gene for dhps , the target for sulfadoxine, has not been studied to date.…”

Plasmodium malariae and Plasmodium ovale infections in the China–Myanmar border area

“…Of note, the S49 and S58 in PmDHFR-TS were different from the C50 and C59 in the PfDHFR sequence. Mutations N50K, K55E, S58R, S59A, S114N/G and I170M found in other PmDHFR sequences [44, 51] were not observed in the PmDHFR sequences from the China–Myanmar border isolates (Table 4; Additional file 7). …”

“…malariae A15 S49 N50 M54K55Y56 F57 S58 S59 T61 T62 S114 L137K161F168 I170 F232K355EF188271, Pm3 [44] P . malariae –– K –E–– R A– – N/G –SS M – – Mutant type [44, 51]C0400117SNMKYFSSTTSLKFIFKChina (KX672042)M0N00290–––––––––––––––––Myanmar (KX672043)M0N00556A–––––…”

Genetic analysis of the orthologous crt and mdr1 genes in Plasmodium malariae from Thailand and Myanmar

“…Thus, the number of parasites under selective pressure would be low for P. malariae and the likelihood of selecting resistant is lower. However, some of the potential drug resistance markers that have been studied in P. malariae might be under selective pressure such as pmdhfr [ 42 , 43 ], pmdhps [ 44 ], and pmkelch [ 45 ]. Thirdly, although clinical P. malariae infection often presents as co-infection with other human Plasmodium species, there might be a large P. malariae reservoir outside of these patients with co-infection or a reservoir in non-human primates, so that overall P. malariae population has been limited to the selective pressure.…”