Patients that are infected with parasites exhibiting the DHFR triple mutant in combination with a DHPS double mutant at positions 437 and 540 have an even higher risk for early treatment failure than with the triple DHFR mutant alone. Moreover,MLN4924 the laboratory findings that show genotypes containing one or more of these mutations to confer antifolate resistance correlate with the molecular markers associated with resistance to one or multiple drugs in the field. Thus, in vitro studies show that a triple mutant N51I/C59R/S108N increases resistance to pyrimethamine 80 fold and resistance to chlorcycloguanil 40 fold. In clinical settings, P. falciparum strains carrying these same mutations are associated with high rates of treatment failure using sulfadoxine-pyrimethamine but remain treatable by chlorproguanil-dapsone. The addition of a mutation of I164L to the triple mutant leads to chlorcycloguanil resistance in addition to pyrimethamine resistance. Although much is known about the genes and mutations leading to chlorcycloguanil resistance,Nilotinib genetics in malaria parasites remains technically challenging thus limiting the scope of research possible in live parasites. The development of heterologous expression systems for pfdhfr in yeast and bacteria have allowed for more extensive research into antifolate resistance. Using transgenic methods, novel alleles conferring drug resistance to pyrimethamine and chlorcycloguanil have been identified by targeted mutagenesis on pfdhfr, and novel alleles leading to multi-drug resistance against pyrimethamine and chlorcycloguanil have been described. These systems also provide a tractable way to examine evolution, and differences in selection pressures on pfdhfr have been described between the antifolates pyrimethamine and WR99210. More recent studies have sought to understand the evolutionary trajectories that may lead to antifolate drug resistance in this system, focusing on resistance to pyrimethamine. The evolution of pyrimethamine resistance has been modeled at the molecular level in yeast and in a comparable system in bacteria.